Modulation of interleukin 22 receptor expression

ABSTRACT

Compounds, compositions and methods are provided for modulating the expression of interleukin 22 receptor. The compositions comprise oligonucleotides, targeted to nucleic acid encoding interleukin 22 receptor. Methods of using these compounds for modulation of interleukin 22 receptor expression and for diagnosis and treatment of disease associated with expression of interleukin 22 receptor are provided.

FIELD OF THE INVENTION

[0001] The present invention provides compositions and methods formodulating the expression of interleukin 22 receptor. In particular,this invention relates to compounds, particularly oligonucleotidecompounds, which, in preferred embodiments, hybridize with nucleic acidmolecules encoding interleukin 22 receptor. Such compounds are shownherein to modulate the expression of interleukin 22 receptor.

BACKGROUND OF THE INVENTION

[0002] Inflammation is a physiological response to various stimuli suchas infection or trauma that results in release of cytokines bymacrophages and lymphocytes. The inflammatory response is characterizedby increased capillary blood flow and permeability allowing variouscells and fluid to leave the capillaries and enter the affected regionresulting in swelling, redness, heat and pain. The increased capillaryblood flow and permeability enables cellular and humoral components ofthe immune system such as cytokines and other mediators to enter theaffected area and assist in the removal of bacteria and repair ofconnective tissues (Kulmatycki and Jamali, Cytokine, 2001, 14, 1-10).

[0003] There are many classes of cytokines such as chemokines,interleukins (IL), tumor necrosis factors (TNF), interferons (IFN),hematoproteins, colony stimulating factors as well as neurotrophins andgrowth factors. The interleukins have both inflammatory andanti-inflammatory activities (Kulmatycki and Jamali, Cytokine, 2001, 14,1-10).

[0004] Interleukin 22 was cloned by Xie et al. who also screened membersof the class II cytokine receptor family for the ability to bind thisligand. By searching an EST database for sequences resembling class IIcytokine receptors, the cDNA encoding interleukin 22 receptor (alsoknown as IL22R and CRF2-9) was identified. The predicted 574-amino acidprotein is most related to interleukin 10 receptors A and B. Expressionof interleukin 22 receptor, like that of interleukin 10 receptor A, isrestricted to hematopoietic tissues, while interleukin 10 receptor B isbroadly expressed. This report was the first to show sharing of receptorcomponents in the class II cytokine receptor family, a phenomenon seenin other cytokine receptor families (Xie et al., J. Biol. Chem., 2000,275, 31335-31339).

[0005] Kotenko et al. also identified the interleukin 22 receptorcomplex and determined that it consists of both interleukin 10 receptorB and interleukin 22 receptor, which they referred to as CRF2-9. Theauthors concluded that interleukin 10 receptor B serves as a commonreceptor chain for both interleukin 10 and interleukin 22 (Kotenko etal., Journal of Biological Chemistry, 2001, 276, 2725-2732).

[0006] Nucleic acid sequences encoding interleukin 22 receptor aredisclosed in U.S. Pat. No. 5,965,704 and PCT publications wo 99/46422and WO 01/40467 (Lok et al., 1999; Presnell and Kindsvogel, 2001;Presnell et al., 2001).

[0007] Currently, there are no known therapeutic agents that effectivelyinhibit the synthesis of interleukin 22 receptor. Consequently, thereremains a long felt need for additional agents capable of effectivelyinhibiting interleukin 22 receptor function.

[0008] Antisense technology is emerging as an effective means forreducing the expression of specific gene products and may thereforeprove to be uniquely useful in a number of therapeutic, diagnostic, andresearch applications for the modulation of expression of interleukin 22receptor.

[0009] The present invention provides compositions and methods formodulating expression of interleukin 22 receptor.

SUMMARY OF THE INVENTION

[0010] The present invention is directed to compounds, especiallynucleic acid and nucleic acid-like oligomers, which are targeted to anucleic acid encoding interleukin 22 receptor, and which modulate theexpression of interleukin 22 receptor. Pharmaceutical and othercompositions comprising the compounds of the invention are alsoprovided. Further provided are methods of screening for modulators ofinterleukin 22 receptor and methods of modulating the expression ofinterleukin 22 receptor in cells, tissues or animals comprisingcontacting said cells, tissues or animals with one or more of thecompounds or compositions of the invention. Methods of treating ananimal, particularly a human, suspected of having or being prone to adisease or condition associated with expression of interleukin 22receptor are also set forth herein. Such methods comprise administeringa therapeutically or prophylactically effective amount of one or more ofthe compounds or compositions of the invention to the person in need oftreatment.

DETAILED DESCRIPTION OF THE INVENTION

[0011] A. Overview of the Invention

[0012] The present invention employs compounds, preferablyoligonucleotides and similar species for use in modulating the functionor effect of nucleic acid molecules encoding interleukin 22 receptor.This is accomplished by providing oligonucleotides which specificallyhybridize with one or more nucleic acid molecules encoding interleukin22 receptor. As used herein, the terms “target nucleic acid” and“nucleic acid molecule encoding interleukin 22 receptor” have been usedfor convenience to encompass DNA encoding interleukin 22 receptor, RNA(including pre-mRNA and mRNA or portions thereof) transcribed from suchDNA, and also cDNA derived from such RNA. The hybridization of acompound of this invention with its target nucleic acid is generallyreferred to as “antisense”. Consequently, the preferred mechanismbelieved to be included in the practice of some preferred embodiments ofthe invention is referred to herein as “antisense inhibition.” Suchantisense inhibition is typically based upon hydrogen bonding-basedhybridization of oligonucleotide strands or segments such that at leastone strand or segment is cleaved, degraded, or otherwise renderedinoperable. In this regard, it is presently preferred to target specificnucleic acid molecules and their functions for such antisenseinhibition.

[0013] The functions of DNA to be interfered with can includereplication and transcription. Replication and transcription, forexample, can be from an endogenous cellular template, a vector, aplasmid construct or otherwise. The functions of RNA to be interferedwith can include functions such as translocation of the RNA to a site ofprotein translation, translocation of the RNA to sites within the cellwhich are distant from the site of RNA synthesis, translation of proteinfrom the RNA, splicing of the RNA to yield one or more RNA species, andcatalytic activity or complex formation involving the RNA which may beengaged in or facilitated by the RNA. One preferred result of suchinterference with target nucleic acid function is modulation of theexpression of interleukin 22 receptor. In the context of the presentinvention, “modulation” and “modulation of expression” mean either anincrease (stimulation) or a decrease (inhibition) in the amount orlevels of a nucleic acid molecule encoding the gene, e.g., DNA or RNA.Inhibition is often the preferred form of modulation of expression andmRNA is often a preferred target nucleic acid.

[0014] In the context of this invention, “hybridization” means thepairing of complementary strands of oligomeric compounds. In the presentinvention, the preferred mechanism of pairing involves hydrogen bonding,which may be Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogenbonding, between complementary nucleoside or nucleotide bases(nucleobases) of the strands of oligomeric compounds. For example,adenine and thymine are complementary nucleobases which pair through theformation of hydrogen bonds. Hybridization can occur under varyingcircumstances.

[0015] An antisense compound is specifically hybridizable when bindingof the compound to the target nucleic acid interferes with the normalfunction of the target nucleic acid to cause a loss of activity, andthere is a sufficient degree of complementarity to avoid non-specificbinding of the antisense compound to non-target nucleic acid sequencesunder conditions in which specific binding is desired, i.e., underphysiological conditions in the case of in vivo assays or therapeutictreatment, and under conditions in which assays are performed in thecase of in vitro assays.

[0016] In the present invention the phrase “stringent hybridizationconditions” or “stringent conditions” refers to conditions under which acompound of the invention will hybridize to its target sequence, but toa minimal number of other sequences. Stringent conditions aresequence-dependent and will be different in different circumstances andin the context of this invention, “stringent conditions” under whicholigomeric compounds hybridize to a target sequence are determined bythe nature and composition of the oligomeric compounds and the assays inwhich they are being investigated.

[0017] “Complementary,” as used herein, refers to the capacity forprecise pairing between two nucleobases of an oligomeric compound. Forexample, if a nucleobase at a certain position of an oligonucleotide (anoligomeric compound), is capable of hydrogen bonding with a nucleobaseat a certain position of a target nucleic acid, said target nucleic acidbeing a DNA, RNA, or oligonucleotide molecule, then the position ofhydrogen bonding between the oligonucleotide and the target nucleic acidis considered to be a complementary position. The oligonucleotide andthe further DNA, RNA, or oligonucleotide molecule are complementary toeach other when a sufficient number of complementary positions in eachmolecule are occupied by nucleobases which can hydrogen bond with eachother. Thus, “specifically hybridizable” and “complementary” are termswhich are used to indicate a sufficient degree of precise pairing orcomplementarity over a sufficient number of nucleobases such that stableand specific binding occurs between the oligonucleotide and a targetnucleic acid.

[0018] It is understood in the art that the sequence of an antisensecompound need not be 100% complementary to that of its target nucleicacid to be specifically hybridizable. Moreover, an oligonucleotide mayhybridize over one or more segments such that intervening or adjacentsegments are not involved in the hybridization event (e.g., a loopstructure or hairpin structure). It is preferred that the antisensecompounds of the present invention comprise at least 70% sequencecomplementarity to a target region within the target nucleic acid, morepreferably that they comprise 90% sequence complementarity and even morepreferably comprise 95% sequence complementarity to the target regionwithin the target nucleic acid sequence to which they are targeted. Forexample, an antisense compound in which 18 of 20 nucleobases of theantisense compound are complementary to a target region, and wouldtherefore specifically hybridize, would represent 90 percentcomplementarity. In this example, the remaining noncomplementarynucleobases may be clustered or interspersed with complementarynucleobases and need not be contiguous to each other or to complementarynucleobases. As such, an antisense compound which is 18 nucleobases inlength having 4 (four) noncomplementary nucleobases which are flanked bytwo regions of complete complementarity with the target nucleic acidwould have 77.8% overall complementarity with the target nucleic acidand would thus fall within the scope of the present invention. Percentcomplementarity of an antisense compound with a region of a targetnucleic acid can be determined routinely using BLAST programs (basiclocal alignment search tools) and PowerBLAST programs known in the art(Altschul et al., J. Mol. Biol., 1990, 215, 403-410; Zhang and Madden,Genome Res., 1997, 7, 649-656).

[0019] B. Compounds of the Invention

[0020] According to the present invention, compounds include antisenseoligomeric compounds, antisense oligonucleotides, ribozymes, externalguide sequence (EGS) oligonucleotides, alternate splicers, primers,probes, and other oligomeric compounds which hybridize to at least aportion of the target nucleic acid. As such, these compounds may beintroduced in the form of single-stranded, double-stranded, circular orhairpin oligomeric compounds and may contain structural elements such asinternal or terminal bulges or loops. Once introduced to a system, thecompounds of the invention may elicit the action of one or more enzymesor structural proteins to effect modification of the target nucleicacid. One non-limiting example of such an enzyme is RNAse H, a cellularendonuclease which cleaves the RNA strand of an RNA:DNA duplex. It isknown in the art that single-stranded antisense compounds which are“DNA-like” elicit RNAse H. Activation of RNase H, therefore, results incleavage of the RNA target, thereby greatly enhancing the efficiency ofoligonucleotide-mediated inhibition of gene expression. Similar roleshave been postulated for other ribonucleases such as those in the RNaseIII and ribonuclease L family of enzymes.

[0021] While the preferred form of antisense compound is asingle-stranded antisense oligonucleotide, in many species theintroduction of double-stranded structures, such as double-stranded RNA(dsRNA) molecules, has been shown to induce potent and specificantisense-mediated reduction of the function of a gene or its associatedgene products. This phenomenon occurs in both plants and animals and isbelieved to have an evolutionary connection to viral defense andtransposon silencing.

[0022] The first evidence that dsRNA could lead to gene silencing inanimals came in 1995 from work in the nematode, Caenorhabditis elegans(Guo and Kempheus, Cell, 1995, 81, 611-620). Montgomery et al. haveshown that the primary interference effects of dsRNA areposttranscriptional (Montgomery et al., Proc. Natl. Acad. Sci. USA,1998, 95, 15502-15507). The posttranscriptional antisense mechanismdefined in Caenorhabditis elegans resulting from exposure todouble-stranded RNA (dsRNA) has since been designated RNA interference(RNAi). This term has been generalized to mean antisense-mediated genesilencing involving the introduction of dsRNA leading to thesequence-specific reduction of endogenous targeted mRNA levels (Fire etal., Nature, 1998, 391, 806-811). Recently, it has been shown that itis, in fact, the single-stranded RNA oligomers of antisense polarity ofthe dsRNAs which are the potent inducers of RNAi (Tijsterman et al.,Science, 2002, 295, 694-697).

[0023] In the context of this invention, the term “oligomeric compound”refers to a polymer or oligomer comprising a plurality of monomericunits. In the context of this invention, the term “oligonucleotide”refers to an oligomer or polymer of ribonucleic acid (RNA) ordeoxyribonucleic acid (DNA) or mimetics, chimeras, analogs and homologsthereof. This term includes oligonucleotides composed of naturallyoccurring nucleobases, sugars and covalent internucleoside (backbone)linkages as well as oligonucleotides having non-naturally occurringportions which function similarly. Such modified or substitutedoligonucleotides are often preferred over native forms because ofdesirable properties such as, for example, enhanced cellular uptake,enhanced affinity for a target nucleic acid and increased stability inthe presence of nucleases.

[0024] While oligonucleotides are a preferred form of the compounds ofthis invention, the present invention comprehends other families ofcompounds as well, including but not limited to oligonucleotide analogsand mimetics such as those described herein.

[0025] The compounds in accordance with this invention preferablycomprise from about 8 to about 80 nucleobases (i.e. from about 8 toabout 80 linked nucleosides). One of ordinary skill in the art willappreciate that the invention embodies compounds of 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48,49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66,67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, or 80 nucleobases inlength.

[0026] In one preferred embodiment, the compounds of the invention are12 to 50 nucleobases in length. One having ordinary skill in the artwill appreciate that this embodies compounds of 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35,36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50nucleobases in length.

[0027] In another preferred embodiment, the compounds of the inventionare 15 to 30 nucleobases in length. One having ordinary skill in the artwill appreciate that this embodies compounds of 15, 16, 17, 18, 19, 20,21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleobases in length.

[0028] Particularly preferred compounds are oligonucleotides from about12 to about 50 nucleobases, even more preferably those comprising fromabout 15 to about 30 nucleobases.

[0029] Antisense compounds 8-80 nucleobases in length comprising astretch of at least eight (8) consecutive nucleobases selected fromwithin the illustrative antisense compounds are considered to besuitable antisense compounds as well.

[0030] Exemplary preferred antisense compounds include oligonucleotidesequences that comprise at least the 8 consecutive nucleobases from the5′-terminus of one of the illustrative preferred antisense compounds(the remaining nucleobases being a consecutive stretch of the sameoligonucleotide beginning immediately upstream of the 5′-terminus of theantisense compound which is specifically hybridizable to the targetnucleic acid and continuing until the oligonucleotide contains about 8to about 80 nucleobases). Similarly preferred antisense compounds arerepresented by oligonucleotide sequences that comprise at least the 8consecutive nucleobases from the 3′-terminus of one of the illustrativepreferred antisense compounds (the remaining nucleobases being aconsecutive stretch of the same oligonucleotide beginning immediatelydownstream of the 3′-terminus of the antisense compound which isspecifically hybridizable to the target nucleic acid and continuinguntil the oligonucleotide contains about 8 to about 80 nucleobases). Onehaving skill in the art armed with the preferred antisense compoundsillustrated herein will be able, without undue experimentation, toidentify further preferred antisense compounds.

[0031] C. Targets of the Invention

[0032] “Targeting” an antisense compound to a particular nucleic acidmolecule, in the context of this invention, can be a multistep process.The process usually begins with the identification of a target nucleicacid whose function is to be modulated. This target nucleic acid may be,for example, a cellular gene (or mRNA transcribed from the gene) whoseexpression is associated with a particular disorder or disease state, ora nucleic acid molecule from an infectious agent. In the presentinvention, the target nucleic acid encodes interleukin 22 receptor.

[0033] The targeting process usually also includes determination of atleast one target region, segment, or site within the target nucleic acidfor the antisense interaction to occur such that the desired effect,e.g., modulation of expression, will result. Within the context of thepresent invention, the term “region” is defined as a portion of thetarget nucleic acid having at least one identifiable structure,function, or characteristic. Within regions of target nucleic acids aresegments. “Segments” are defined as smaller or sub-portions of regionswithin a target nucleic acid. “Sites,” as used in the present invention,are defined as positions within a target nucleic acid.

[0034] Since, as is known in the art, the translation initiation codonis typically 5′-AUG (in transcribed mRNA molecules; 5′-ATG in thecorresponding DNA molecule), the translation initiation codon is alsoreferred to as the “AUG codon,” the “start codon” or the “AUG startcodon”. A minority of genes have a translation initiation codon havingthe RNA sequence 5′-GUG, 5′-UUG or 5′-CUG, and 5′-AUA, 5′-ACG and 5′-CUGhave been shown to function in vivo. Thus, the terms “translationinitiation codon” and “start codon” can encompass many codon sequences,even though the initiator amino acid in each instance is typicallymethionine (in eukaryotes) or formylmethionine (in prokaryotes). It isalso known in the art that eukaryotic and prokaryotic genes may have twoor more alternative start codons, any one of which may be preferentiallyutilized for translation initiation in a particular cell type or tissue,or under a particular set of conditions. In the context of theinvention, “start codon” and “translation initiation codon” refer to thecodon or codons that are used in vivo to initiate translation of an mRNAtranscribed from a gene encoding interleukin 22 receptor, regardless ofthe sequence(s) of such codons. It is also known in the art that atranslation termination codon (or “stop codon”) of a gene may have oneof three sequences, i.e., 5′-UAA, 5′-UAG and 5′-UGA (the correspondingDNA sequences are 5′-TAA, 5′-TAG and 5′-TGA, respectively).

[0035] The terms “start codon region” and “translation initiation codonregion” refer to a portion of such an mRNA or gene that encompasses fromabout 25 to about 50 contiguous nucleotides in either direction (i.e.,51 or 3′) from a translation initiation codon. Similarly, the terms“stop codon region” and “translation termination codon region” refer toa portion of such an mRNA or gene that encompasses from about 25 toabout 50 contiguous nucleotides in either direction (i.e., 5′ or 3′)from a translation termination codon. Consequently, the “start codonregion” (or “translation initiation codon region”) and the “stop codonregion” (or “translation termination codon region”) are all regionswhich may be targeted effectively with the antisense compounds of thepresent invention.

[0036] The open reading frame (ORF) or “coding region,” which is knownin the art to refer to the region between the translation initiationcodon and the translation termination codon, is also a region which maybe targeted effectively. Within the context of the present invention, apreferred region is the intragenic region encompassing the translationinitiation or termination codon of the open reading frame (ORF) of agene.

[0037] Other target regions include the 5′ untranslated region (5′UTR),known in the art to refer to the portion of an mRNA in the 5′ directionfrom the translation initiation codon, and thus including nucleotidesbetween the 5′ cap site and the translation initiation codon of an mRNA(or corresponding nucleotides on the gene), and the 3′ untranslatedregion (3′UTR), known in the art to refer to the portion of an mRNA inthe 3′ direction from the translation termination codon, and thusincluding nucleotides between the translation termination codon and 3′end of an mRNA (or corresponding nucleotides on the gene). The 5′ capsite of an mRNA comprises an N7-methylated guanosine residue joined tothe 5′-most residue of the mRNA via a 5′-5′ triphosphate linkage. The 5′cap region of an mRNA is considered to include the 5′ cap structureitself as well as the first 50 nucleotides adjacent to the cap site. Itis also preferred to target the 5′ cap region.

[0038] Although some eukaryotic mRNA transcripts are directlytranslated, many contain one or more regions, known as “introns,” whichare excised from a transcript before it is translated. The remaining(and therefore translated) regions are known as “exons” and are splicedtogether to form a continuous mRNA sequence. Targeting splice sites,i.e., intron-exon junctions or exon-intron junctions, may also beparticularly useful in situations where aberrant splicing is implicatedin disease, or where an overproduction of a particular splice product isimplicated in disease. Aberrant fusion junctions due to rearrangementsor deletions are also preferred target sites. mRNA transcripts producedvia the process of splicing of two (or more) mRNAs from different genesources are known as “fusion transcripts”. It is also known that intronscan be effectively targeted using antisense compounds targeted to, forexample, DNA or prem-RNA.

[0039] It is also known in the art that alternative RNA transcripts canbe produced from the same genomic region of DNA. These alternativetranscripts are generally known as “variants”. More specifically,“pre-mRNA variants” are transcripts produced from the same genomic DNAthat differ from other transcripts produced from the same genomic DNA ineither their start or stop position and contain both intronic and exonicsequence.

[0040] Upon excision of one or more exon or intron regions, or portionsthereof during splicing, pre-mRNA variants produce smaller “mRNAvariants”. Consequently, mRNA variants are processed pre-mRNA variantsand each unique pre-mRNA variant must always produce a unique mRNAvariant as a result of splicing. These mRNA variants are also known as“alternative splice variants”. If no splicing of the pre-mRNA variantoccurs then the pre-mRNA variant is identical to the mRNA variant.

[0041] It is also known in the art that variants can be produced throughthe use of alternative signals to start or stop transcription and thatpre-mRNAs and mRNAs can possess more that one start codon or stop codon.Variants that originate from a pre-mRNA or mRNA that use alternativestart codons are known as “alternative start variants” of that prem-RNAor mRNA. Those transcripts that use an alternative stop codon are knownas “alternative stop variants” of that prem-RNA or mRNA. One specifictype of alternative stop variant is the “polyA variant” in which themultiple transcripts produced result from the alternative selection ofone of the “polyA stop signals” by the transcription machinery, therebyproducing transcripts that terminate at unique polyA sites. Within thecontext of the invention, the types of variants described herein arealso preferred target nucleic acids.

[0042] The locations on the target nucleic acid to which the preferredantisense compounds hybridize are hereinbelow referred to as “preferredtarget segments.” As used herein the term “preferred target segment” isdefined as at least an 8-nucleobase portion of a target region to whichan active antisense compound is targeted. While not wishing to be boundby theory, it is presently believed that these target segments representportions of the target nucleic acid which are accessible forhybridization.

[0043] While the specific sequences of certain preferred target segmentsare set forth herein, one of skill in the art will recognize that theseserve to illustrate and describe particular embodiments within the scopeof the present invention. Additional preferred target segments may beidentified by one having ordinary skill.

[0044] Target segments 8-80 nucleobases in length comprising a stretchof at least eight (8) consecutive nucleobases selected from within theillustrative preferred target segments are considered to be suitable fortargeting as well.

[0045] Target segments can include DNA or RNA sequences that comprise atleast the 8 consecutive nucleobases from the 5′-terminus of one of theillustrative preferred target segments (the remaining nucleobases beinga consecutive stretch of the same DNA or RNA beginning immediatelyupstream of the 5′-terminus of the target segment and continuing untilthe DNA or RNA contains about 8 to about 80 nucleobases). Similarlypreferred target segments are-represented by DNA or RNA sequences thatcomprise at least the 8 consecutive nucleobases from the 3′-terminus ofone of the illustrative preferred target segments (the remainingnucleobases being a consecutive stretch of the same DNA or RNA beginningimmediately downstream of the 3′-terminus of the target segment andcontinuing until the DNA or RNA contains about 8 to about 80nucleobases). One having skill in the art armed with the preferredtarget segments illustrated herein will be able, without undueexperimentation, to identify further preferred target segments.

[0046] Once one or more target regions, segments or sites have beenidentified, antisense compounds are chosen which are sufficientlycomplementary to the target, i.e., hybridize sufficiently well and withsufficient specificity, to give the desired effect.

[0047] D. Screening and Target Validation

[0048] In a further embodiment, the “preferred target segments”identified herein may be employed in a screen for additional compoundsthat modulate the expression of interleukin 22 receptor. “Modulators”are those compounds that decrease or increase the expression of anucleic acid molecule encoding interleukin 22 receptor and whichcomprise at least an 8-nucleobase portion which is complementary to apreferred target segment. The screening method comprises the steps ofcontacting a preferred target segment of a nucleic acid moleculeencoding interleukin 22 receptor with one or more candidate modulators,and selecting for one or more candidate modulators which decrease orincrease the expression of a nucleic acid molecule encoding interleukin22 receptor. Once it is shown that the candidate modulator or modulatorsare capable of modulating (e.g. either decreasing or increasing) theexpression of a nucleic acid molecule encoding interleukin 22 receptor,the modulator may then be employed in further investigative studies ofthe function of interleukin 22 receptor, or for use as a research,diagnostic, or therapeutic agent in accordance with the presentinvention.

[0049] The preferred target segments of the present invention may bealso be combined with their respective complementary antisense compoundsof the present invention to form stabilized double-stranded (duplexed)oligonucleotides.

[0050] Such double stranded oligonucleotide moieties have been shown inthe art to modulate target expression and regulate translation as wellas RNA processsing via an antisense mechanism. Moreover, thedouble-stranded moieties may be subject to chemical modifications (Fireet al., Nature, 1998, 391, 806-811; Timmons and Fire, Nature 1998, 395,854; Timmons et al., Gene, 2001, 263, 103-112; Tabara et al., Science,1998, 282, 430-431; Montgomery et al., Proc. Natl. Acad. Sci. USA, 1998,95, 15502-15507; Tuschl et al., Genes Dev., 1999, 13, 3191-3197;Elbashir et al., Nature, 2001, 411, 494-498; Elbashir et al., Genes Dev.2001, 15, 188-200). For example, such double-stranded moieties have beenshown to inhibit the target by the classical hybridization of antisensestrand of the duplex to the target, thereby triggering enzymaticdegradation of the target (Tijsterman et al., Science, 2002, 295,694-697).

[0051] The compounds of the present invention can also be applied in theareas of drug discovery and target validation. The present inventioncomprehends the use of the compounds and preferred target segmentsidentified herein in drug discovery efforts to elucidate relationshipsthat exist between interleukin 22 receptor and a disease state,phenotype, or condition. These methods include detecting or modulatinginterleukin 22 receptor comprising contacting a sample, tissue, cell, ororganism with the compounds of the present invention, measuring thenucleic acid or protein level of interleukin 22 receptor and/or arelated phenotypic or chemical endpoint at some time after treatment,and optionally comparing the measured value to a non-treated sample orsample treated with a further compound of the invention. These methodscan also be performed in parallel or in combination with otherexperiments to determine the function of unknown genes for the processof target validation or to determine the validity of a particular geneproduct as a target for treatment or prevention of a particular disease,condition, or phenotype.

[0052] E. Kits, Research Reagents, Diagnostics, and Therapeutics

[0053] The compounds of the present invention can be utilized fordiagnostics, therapeutics, prophylaxis and as research reagents andkits. Furthermore, antisense oligonucleotides, which are able to inhibitgene expression with exquisite specificity, are often used by those ofordinary skill to elucidate the function of particular genes or todistinguish between functions of various members of a biologicalpathway.

[0054] For use in kits and diagnostics, the compounds of the presentinvention, either alone or in combination with other compounds ortherapeutics, can be used as tools in differential and/or combinatorialanalyses to elucidate expression patterns of a portion or the entirecomplement of genes expressed within cells and tissues.

[0055] As one nonlimiting example, expression patterns within cells ortissues treated with one or more antisense compounds are compared tocontrol cells or tissues not treated with antisense compounds and thepatterns produced are analyzed for differential levels of geneexpression as they pertain, for example, to disease association,signaling pathway, cellular localization, expression level, size,structure or function of the genes examined. These analyses can beperformed on stimulated or unstimulated cells and in the presence orabsence of other compounds which affect expression patterns.

[0056] Examples of methods of gene expression analysis known in the artinclude DNA arrays or microarrays (Brazma and Vilo, FEBS Lett., 2000,480, 17-24; Celis, et al., FEBS Lett., 2000, 480, 2-16), SAGE (serialanalysis of gene expression)(Madden, et al., Drug Discov. Today, 2000,5, 415-425), READS (restriction enzyme amplification of digested cDNAs)(Prashar and Weissman, Methods Enzymol., 1999, 303, 258-72), TOGA (totalgene expression analysis) (Sutcliffe, et al., Proc. Natl. Acad. Sci.U.S. A., 2000, 97, 1976-81), protein arrays and proteomics (Celis, etal., FEBS Lett., 2000, 480, 2-16; Jungblut, et al., Electrophoresis,1999, 20, 2100-10), expressed sequence tag (EST) sequencing (Celis, etal., FEBS Lett., 2000, 480, 2-16; Larsson, et al., J. Biotechnol., 2000,80, 143-57), subtractive RNA fingerprinting (SURF) (Fuchs, et al., Anal.Biochem., 2000, 286, 91-98; Larson, et al., Cytometry, 2000, 41,203-208), subtractive cloning, differential display (DD) (Jurecic andBelmont, Curr. Opin. Microbiol., 2000, 3, 316-21), comparative genomichybridization (Carulli, et al., J. Cell Biochem. Suppl., 1998, 31,286-96), FISH (fluorescent in situ hybridization) techniques (Going andGusterson, Eur. J. Cancer, 1999, 35, 1895-904) and mass spectrometrymethods (To, Comb. Chem. High Throughput Screen, 2000, 3, 235-41).

[0057] The compounds of the invention are useful for research anddiagnostics, because these compounds hybridize to nucleic acids encodinginterleukin 22 receptor. For example, oligonucleotides that are shown tohybridize with such efficiency and under such conditions as disclosedherein as to be effective interleukin 22 receptor inhibitors will alsobe effective primers or probes under conditions favoring geneamplification or detection, respectively. These primers and probes areuseful in methods requiring the specific detection of nucleic acidmolecules encoding interleukin 22 receptor and in the amplification ofsaid nucleic acid molecules for detection or for use in further studiesof interleukin 22 receptor. Hybridization of the antisenseoligonucleotides, particularly the primers and probes, of the inventionwith a nucleic acid encoding interleukin 22 receptor can be detected bymeans known in the art. Such means may include conjugation of an enzymeto the oligonucleotide, radiolabelling of the oligonucleotide or anyother suitable detection means. Kits using such detection means fordetecting the level of interleukin 22 receptor in a sample may also beprepared.

[0058] The specificity and sensitivity of antisense is also harnessed bythose of skill in the art for therapeutic uses. Antisense compounds havebeen employed as therapeutic moieties in the treatment of disease statesin animals, including humans. Antisense oligonucleotide drugs, includingribozymes, have been safely and effectively administered to humans andnumerous clinical trials are presently underway. It is thus establishedthat antisense compounds can be useful therapeutic modalities that canbe configured to be useful in treatment regimes for the treatment ofcells, tissues and animals, especially humans.

[0059] For therapeutics, an animal, preferably a human, suspected ofhaving a disease or disorder which can be treated by modulating theexpression of interleukin 22 receptor is treated by administeringantisense compounds in accordance with this invention. For example, inone non-limiting embodiment, the methods comprise the step ofadministering to the animal in need of treatment, a therapeuticallyeffective amount of a interleukin 22 receptor inhibitor. The interleukin22 receptor inhibitors of the present invention effectively inhibit theactivity of the interleukin 22 receptor protein or inhibit theexpression of the interleukin 22 receptor protein. In one embodiment,the activity or expression of interleukin 22 receptor in an animal isinhibited by about 10%. Preferably, the activity or expression ofinterleukin 22 receptor in an animal is inhibited by about 30%. Morepreferably, the activity or expression of interleukin 22 receptor in ananimal is inhibited by 50% or more.

[0060] For example, the reduction of the expression of interleukin 22receptor may be measured in serum, adipose tissue, liver or any otherbody fluid, tissue or organ of the animal. Preferably, the cellscontained within said fluids, tissues or organs being analyzed contain anucleic acid molecule encoding interleukin 22 receptor protein and/orthe interleukin 22 receptor protein itself.

[0061] The compounds of the invention can be utilized in pharmaceuticalcompositions by adding an effective amount of a compound to a suitablepharmaceutically acceptable diluent or carrier. Use of the compounds andmethods of the invention may also be useful prophylactically.

[0062] F. Modifications

[0063] As is known in the art, a nucleoside is a base-sugar combination.The base portion of the nucleoside is normally a heterocyclic base. Thetwo most common classes of such heterocyclic bases are the purines andthe pyrimidines. Nucleotides are nucleosides that further include aphosphate group covalently linked to the sugar portion of thenucleoside. For those nucleosides that include a pentofuranosyl sugar,the phosphate group can be linked to either the 2′, 3′ or 5′ hydroxylmoiety of the sugar. In forming oligonucleotides, the phosphate groupscovalently link adjacent nucleosides to one another to form a linearpolymeric compound. In turn, the respective ends of this linearpolymeric compound can be further joined to form a circular compound,however, linear compounds are generally preferred. In addition, linearcompounds may have internal nucleobase complementarity and may thereforefold in a manner as to produce a fully or partially double-strandedcompound. Within oligonucleotides, the phosphate groups are commonlyreferred to as forming the internucleoside backbone of theoligonucleotide. The normal linkage or backbone of RNA and DNA is a 3′to 5′ phosphodiester linkage.

[0064] Modified Internucleoside Linkages (Backbones)

[0065] Specific examples of preferred antisense compounds useful in thisinvention include oligonucleotides containing modified backbones ornon-natural internucleoside linkages. As defined in this specification,oligonucleotides having modified backbones include those that retain aphosphorus atom in the backbone and those that do not have a phosphorusatom in the backbone. For the purposes of this specification, and assometimes referenced in the art, modified oligonucleotides that do nothave a phosphorus atom in their internucleoside backbone can also beconsidered to be oligonucleosides.

[0066] Preferred modified oligonucleotide backbones containing aphosphorus atom therein include, for example, phosphorothioates, chiralphosphorothioates, phosphorodithioates, phosphotriesters,aminoalkylphosphotriesters, methyl and other alkyl phosphonatesincluding 3′-alkylene phosphonates, 5′-alkylene phosphonates and chiralphosphonates, phosphinates, phosphoramidates including 3′-aminophosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates,thionoalkylphosphonates, thionoalkylphosphotriesters, selenophosphatesand boranophosphates having normal 3′-5′ linkages, 2′-5′ linked analogsof these, and those having inverted polarity wherein one or moreinternucleotide linkages is a 3′ to 3′, 5′ to 5′ or 2′ to 2′ linkage.Preferred oligonucleotides having inverted polarity comprise a single 3′to 3′ linkage at the 3′-most internucleotide linkage i.e. a singleinverted nucleoside residue which may be abasic (the nucleobase ismissing or has a hydroxyl group in place thereof). Various salts, mixedsalts and free acid forms are also included.

[0067] Representative United States patents that teach the preparationof the above phosphorus-containing linkages include, but are not limitedto, U.S. Pat. Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243;5,177,196; 5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717;5,321,131; 5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677;5,476,925; 5,519,126; 5,536,821; 5,541,306; 5,550,111; 5,563,253;5,571,799; 5,587,361; 5,194,599; 5,565,555; 5,527,899; 5,721,218;5,672,697 and 5,625,050, certain of which are commonly owned with thisapplication, and each of which is herein incorporated by reference.

[0068] Preferred modified oligonucleotide backbones that do not includea phosphorus atom therein have backbones that are formed by short chainalkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkylor cycloalkyl internucleoside linkages, or one or more short chainheteroatomic or heterocyclic internucleoside linkages. These includethose having morpholino linkages (formed in part from the sugar portionof a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfonebackbones; formacetyl and thioformacetyl backbones; methylene formacetyland thioformacetyl backbones; riboacetyl backbones; alkene containingbackbones; sulfamate backbones; methyleneimino and methylenehydrazinobackbones; sulfonate and sulfonamide backbones; amide backbones; andothers having mixed N, O, S and CH₂ component parts.

[0069] Representative United States patents that teach the preparationof the above oligonucleosides include, but are not limited to, U.S. Pat.Nos. 5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033;5,264,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967;5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,610,289;5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312;5,633,360; 5,677,437; 5,792,608; 5,646,269 and 5,677,439, certain ofwhich are commonly owned with this application, and each of which isherein incorporated by reference.

[0070] Modified Sugar and Internucleoside Linkages-Mimetics

[0071] In other preferred oligonucleotide mimetics, both the sugar andthe internucleoside linkage (i.e. the backbone), of the nucleotide unitsare replaced with novel groups. The nucleobase units are maintained forhybridization with an appropriate target nucleic acid. One suchcompound, an oligonucleotide mimetic that has been shown to haveexcellent hybridization properties, is referred to as a peptide nucleicacid (PNA). In PNA compounds, the sugar-backbone of an oligonucleotideis replaced with an amide containing backbone, in particular anaminoethylglycine backbone. The nucleobases are retained and are bounddirectly or indirectly to aza nitrogen atoms of the amide portion of thebackbone. Representative United States patents that teach thepreparation of PNA compounds include, but are not limited to, U.S. Pat.Nos. 5,539,082; 5,714,331; and 5,719,262, each of which is hereinincorporated by reference. Further teaching of PNA compounds can befound in Nielsen et al., Science, 1991, 254, 1497-1500.

[0072] Preferred embodiments of the invention are oligonucleotides withphosphorothioate backbones and oligonucleosides with heteroatombackbones, and in particular —CH₂—NH—O—CH₂—, —CH₂—N(CH₃)—O—CH₂— [knownas a methylene (methylimino) or MMI backbone], —CH₂—O—N(CH₃)—CH₂—,—CH₂N(CH₃)—N(CH₃)—CH₂— and —O—N(CH₃)—CH₂—CH₂— [wherein the nativephosphodiester backbone is represented as —O—P—O—CH₂—] of the abovereferenced U.S. Pat. No. 5,489,677, and the amide backbones of the abovereferenced U.S. Pat. No. 5,602,240. Also preferred are oligonucleotideshaving morpholino backbone structures of the above-referenced U.S. Pat.No. 5,034,506.

[0073] Modified Sugars

[0074] Modified oligonucleotides may also contain one or moresubstituted sugar moieties. Preferred oligonucleotides comprise one ofthe following at the 2′ position: OH; F; O-, S-, or N-alkyl; O-, S-, orN-alkenyl; O-, S- or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl,alkenyl and alkynyl may be substituted or unsubstituted C₁ to C₁₀ alkylor C₂ to C₁₀ alkenyl and alkynyl. Particularly preferred areO[(CH₂)_(n)O]_(m)CH₃, O(CH₂)_(n)OCH₃, O(CH₂)_(n)NH₂, O(CH₂)CH₃,O(CH₂)_(n)ONH₂, and O(CH₂)_(n)ON[(CH₂)_(n)CH₃]₂, where n and m are from1 to about 10. Other preferred oligonucleotides comprise one of thefollowing at the 2′ position: C₁ to C₁₀ lower alkyl, substituted loweralkyl, alkenyl, alkynyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH,SCH₃, OCN, Cl, Br, CN, CF₃, OCF₃, SOCH₃, SO₂CH₃, ONO₂, NO₂, N₃, NH₂,heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino,substituted silyl, an RNA cleaving group, a reporter group, anintercalator, a group for improving the pharmacokinetic properties of anoligonucleotide, or a group for improving the pharmacodynamic propertiesof an oligonucleotide, and other substituents having similar properties.A preferred modification includes 2′-methoxyethoxy (2′-O—CH₂CH₂OCH₃,also known as 2′-O-(2-methoxyethyl) or 2′-MOE) (Martin et al., Helv.Chim. Acta, 1995, 78, 486-504) i.e., an alkoxyalkoxy group. A furtherpreferred modification includes 2′-dimethylaminooxyethoxy, i.e., aO(CH₂)₂ON(CH₃)₂ group, also known as 2′-DMAOE, as described in exampleshereinbelow, and 2′-dimethylaminoethoxyethoxy (also known in the art as2′-O-dimethyl-amino-ethoxy-ethyl or 2′-DMAEOE), i.e.,2′-O—CH₂—OCH₂—N(CH₃)₂, also described in examples hereinbelow.

[0075] Other preferred modifications include 2′-methoxy (2′-O—CH₃),2′-aminopropoxy (2′-OCH₂CH₂CH₂NH₂), 2′-allyl (2′-CH₂—CH═CH₂), 2′-O-allyl(2′-O—CH₂—CH═CH₂) and 2′-fluoro (2′-F). The 2′-modification may be inthe arabino (up) position or ribo (down) position. A preferred2′-arabino modification is 2′-F. Similar modifications may also be madeat other positions on the oligonucleotide, particularly the 3′ positionof the sugar on the 3′ terminal nucleotide or in 2′-5′ linkedoligonucleotides and the 5′ position of 5′ terminal nucleotideoligonucleotides may also have sugar mimetics such as cyclobutylmoieties in place of the pentofuranosyl sugar. Representative UnitedStates patents that teach the preparation of such modified sugarstructures include, but are not limited to, U.S. Pat. Nos. 4,981,957;5,118,800; 5,319,080; 5,359,044; 5,393,878; 5,446,137; 5,466,786;5,514,785; 5,519,134; 5,567,811; 5,576,427; 5,591,722; 5,597,909;5,610,300; 5,627,053; 5,639,873; 5,646,265; 5,658,873; 5,670,633;5,792,747; and 5,700,920, certain of which are commonly owned with theinstant application, and each of which is herein incorporated byreference in its entirety.

[0076] A further preferred modification of the sugar includes LockedNucleic Acids (LNAs) in which the 2′-hydroxyl group is linked to the 3′or 4′ carbon atom of the sugar ring, thereby forming a bicyclic sugarmoiety. The linkage is preferably a methelyne (—CH₂—)_(n) group bridgingthe 2′ oxygen atom and the 4′ carbon atom wherein n is 1 or 2. LNAs andpreparation thereof are described in WO 98/39352 and WO 99/14226.

[0077] Natural and Modified Nucleobases

[0078] Oligonucleotides may also include nucleobase (often referred toin the art simply as “base”) modifications or substitutions. As usedherein, “unmodified” or “natural” nucleobases include the purine basesadenine (A) and guanine (G), and the pyrimidine bases thymine (T),cytosine (C) and uracil (U). Modified nucleobases include othersynthetic and natural nucleobases such as 5-methylcytosine (5-me-C),5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine,6-methyl and other alkyl derivatives of adenine and guanine, 2-propyland other alkyl derivatives of adenine and guanine, 2-thiouracil,2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl(—C═—C—CH₃) uracil and cytosine and other alkynyl derivatives ofpyrimidine bases, 6-azo uracil, cytosine and thymine, 5-uracil(pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl,8-hydroxyl and other 8-substituted adenines and guanines, 5-haloparticularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracilsand cytosines, 7-methylguanine and 7-methyladenine, 2-F-adenine,2-amino-adenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and7-deazaadenine and 3-deazaguanine and 3-deazaadenine. Further modifiednucleobases include tricyclic pyrimidines such as phenoxazinecytidine(1H-pyrimido[5,4-b][1,4]benzoxazin-2(3H)-one), phenothiazinecytidine (1H-pyrimido[5,4-b][1,4]benzothiazin-2(3H)-one), G-clamps suchas a substituted phenoxazine cytidine (e.g.9-(2-aminoethoxy)-H-pyrimido[5,4-b][1,4]benzoxazin-2(3H)-one), carbazolecytidine (2H-pyrimido[4,5-b]indol-2-one), pyridoindole cytidine(H-pyrido[3′,2′:4,5]pyrrolo[2,3-d]pyrimidin-2-one). Modified nucleobasesmay also include those in which the purine or pyrimidine base isreplaced with other heterocycles, for example 7-deaza-adenine,7-deazaguanosine, 2-aminopyridine and 2-pyridone. Further nucleobasesinclude those disclosed in U.S. Pat. No. 3,687,808, those disclosed inThe Concise Encyclopedia Of Polymer Science And Engineering, pages858-859, Kroschwitz, J. I., ed. John Wiley & Sons, 1990, those disclosedby Englisch et al., Angewandte Chemie, International Edition, 1991, 30,613, and those disclosed by Sanghvi, Y. S., Chapter 15, AntisenseResearch and Applications, pages 289-302, Crooke, S. T. and Lebleu, B.ed., CRC Press, 1993. Certain of these nucleobases are particularlyuseful for increasing the binding affinity of the compounds of theinvention. These include 5-substituted pyrimidines, 6-azapyrimidines andN-2, N-6 and O-6 substituted purines, including 2-aminopropyladenine,5-propynyluracil and 5-propynylcytosine. 5-methylcytosine substitutionshave been shown to increase nucleic acid duplex stability by 0.6-1.2° C.and are presently preferred base substitutions, even more particularlywhen combined with 2′-O-methoxyethyl sugar modifications.

[0079] Representative United States patents that teach the preparationof certain of the above noted modified nucleobases as well as othermodified nucleobases include, but are not limited to, the above notedU.S. Pat. No. 3,687,808, as well as U.S. Pat. Nos. 4,845,205; 5,130,302;5,134,066; 5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,459,255;5,484,908; 5,502,177; 5,525,711; 5,552,540; 5,587,469; 5,594,121,5,596,091; 5,614,617; 5,645,985; 5,830,653; 5,763,588; 6,005,096; and5,681,941, certain of which are commonly owned with the instantapplication, and each of which is herein incorporated by reference, andU.S. Pat. No. 5,750,692, which is commonly owned with the instantapplication and also herein incorporated by reference.

[0080] Conjugates

[0081] Another modification of the oligonucleotides of the inventioninvolves chemically linking to the oligonucleotide one or more moietiesor conjugates which enhance the activity, cellular distribution orcellular uptake of the oligonucleotide. These moieties or conjugates caninclude conjugate groups covalently bound to functional groups such asprimary or secondary hydroxyl groups. Conjugate groups of the inventioninclude intercalators, reporter molecules, polyamines, polyamides,polyethylene glycols, polyethers, groups that enhance thepharmacodynamic properties of oligomers, and groups that enhance thepharmacokinetic properties of oligomers. Typical conjugate groupsinclude cholesterols, lipids, phospholipids, biotin, phenazine, folate,phenanthridine, anthraquinone, acridine, fluoresceins, rhodamines,coumarins, and dyes. Groups that enhance the pharmacodynamic properties,in the context of this invention, include groups that improve uptake,enhance resistance to degradation, and/or strengthen sequence-specifichybridization with the target nucleic acid. Groups that enhance thepharmacokinetic properties, in the context of this invention, includegroups that improve uptake, distribution, metabolism or excretion of thecompounds of the present invention. Representative conjugate groups aredisclosed in International Patent Application PCT/US92/09196, filed Oct.23, 1992, and U.S. Pat. No. 6,287,860, the entire disclosure of whichare incorporated herein by reference. Conjugate moieties include but arenot limited to lipid moieties such as a cholesterol moiety, cholic acid,a thioether, e.g., hexyl-S-tritylthiol, a thiocholesterol, an aliphaticchain, e.g., dodecandiol or undecyl residues, a phospholipid, e.g.,di-hexadecyl-rac-glycerol or triethyl-ammonium1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate, a polyamine or apolyethylene glycol chain, or adamantane acetic acid, a palmityl moiety,or an octadecylamine or hexylamino-carbonyl-oxycholesterol moiety.Oligonucleotides of the invention may also be conjugated to active drugsubstances, for example, aspirin, warfarin, phenylbutazone, ibuprofen,suprofen, fenbufen, ketoprofen, (S)-(+)-pranoprofen, carprofen,dansylsarcosine, 2,3,5-triiodobenzoic acid, flufenamic acid, folinicacid, a benzothiadiazide, chlorothiazide, a diazepine, indomethicin, abarbiturate, a cephalosporin, a sulfa drug, an antidiabetic, anantibacterial or an antibiotic. Oligonucleotide-drug conjugates andtheir preparation are described in U.S. patent application Ser. No.09/334,130 (filed Jun. 15, 1999) which is incorporated herein byreference in its entirety.

[0082] Representative United States patents that teach the preparationof such oligonucleotide conjugates include, but are not limited to, U.S.Pat. Nos. 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313;5,545,730; 5,552,538; 5,578,717, 5,580,731; 5,580,731; 5,591,584;5,109,124; 5,118,802; 5,138,045; 5,414,077; 5,486,603; 5,512,439;5,578,718; 5,608,046; 4,587,044; 4,605,735; 4,667,025; 4,762,779;4,789,737; 4,824,941; 4,835,263; 4,876,335; 4,904,582; 4,958,013;5,082,830; 5,112,963; 5,214,136; 5,082,830; 5,112,963; 5,214,136;5,245,022; 5,254,469; 5,258,506; 5,262,536; 5,272,250; 5,292,873;5,317,098; 5,371,241, 5,391,723; 5,416,203, 5,451,463; 5,510,475;5,512,667; 5,514,785; 5,565,552; 5,567,810; 5,574,142; 5,585,481;5,587,371; 5,595,726; 5,597,696; 5,599,923; 5,599,928 and 5,688,941,certain of which are commonly owned with the instant application, andeach of which is herein incorporated by reference.

[0083] Chimeric Compounds

[0084] It is not necessary for all positions in a given compound to beuniformly modified, and in fact more than one of the aforementionedmodifications may be incorporated in a single compound or even at asingle nucleoside within an oligonucleotide.

[0085] The present invention also includes antisense compounds which arechimeric compounds. “Chimeric” antisense compounds or “chimeras,” in thecontext of this invention, are antisense compounds, particularlyoligonucleotides, which contain two or more chemically distinct regions,each made up of at least one monomer unit, i.e., a nucleotide in thecase of an oligonucleotide compound. These oligonucleotides typicallycontain at least one region wherein the oligonucleotide is modified soas to confer upon the oligonucleotide increased resistance to nucleasedegradation, increased cellular uptake, increased stability and/orincreased binding affinity for the target nucleic acid. An additionalregion of the oligonucleotide may serve as a substrate for enzymescapable of cleaving RNA:DNA or RNA:RNA hybrids. By way of example, RNAseH is a cellular endonuclease which cleaves the RNA strand of an RNA:DNAduplex. Activation of RNase H, therefore, results in cleavage of the RNAtarget, thereby greatly enhancing the efficiency ofoligonucleotide-mediated inhibition of gene expression. The cleavage ofRNA:RNA hybrids can, in like fashion, be accomplished through theactions of endoribonucleases, such as RNAseL which cleaves both cellularand viral RNA. Cleavage of the RNA target can be routinely detected bygel electrophoresis and, if necessary, associated nucleic acidhybridization techniques known in the art.

[0086] Chimeric antisense compounds of the invention may be formed ascomposite structures of two or more oligonucleotides, modifiedoligonucleotides, oligonucleosides and/or oligonucleotide mimetics asdescribed above. Such compounds have also been referred to in the art ashybrids or gapmers. Representative United States patents that teach thepreparation of such hybrid structures include, but are not limited to,U.S. Pat. Nos. 5,013,830; 5,149,797; 5,220,007; 5,256,775; 5,366,878;5,403,711; 5,491,133; 5,565,350; 5,623,065; 5,652,355; 5,652,356; and5,700,922, certain of which are commonly owned with the instantapplication, and each of which is herein incorporated by reference inits entirety.

[0087] G. Formulations

[0088] The compounds of the invention may also be admixed, encapsulated,conjugated or otherwise associated with other molecules, moleculestructures or mixtures of compounds, as for example, liposomes,receptor-targeted molecules, oral, rectal, topical or otherformulations, for assisting in uptake, distribution and/or absorption.Representative United States patents that teach the preparation of suchuptake, distribution and/or absorption-assisting formulations include,but are not limited to, U.S. Pat. Nos. 5,108,921; 5,354,844; 5,416,016;5,459,127; 5,521,291; 5,543,158; 5,547,932; 5,583,020; 5,591,721;4,426,330; 4,534,899; 5,013,556; 5,108,921; 5,213,804; 5,227,170;5,264,221; 5,356,633; 5,395,619; 5,416,016; 5,417,978; 5,462,854;5,469,854; 5,512,295; 5,527,528; 5,534,259; 5,543,152; 5,556,948;5,580,575; and 5,595,756, each of which is herein incorporated byreference.

[0089] The antisense compounds of the invention encompass anypharmaceutically acceptable salts, esters, or salts of such esters, orany other compound which, upon administration to an animal, including ahuman, is capable of providing (directly or indirectly) the biologicallyactive metabolite or residue thereof. Accordingly, for example, thedisclosure is also drawn to prodrugs and pharmaceutically acceptablesalts of the compounds of the invention, pharmaceutically acceptablesalts of such prodrugs, and other bioequivalents.

[0090] The term “prodrug” indicates a therapeutic agent that is preparedin an inactive form that is converted to an active form (i.e., drug)within the body or cells thereof by the action of endogenous enzymes orother chemicals and/or conditions. In particular, prodrug versions ofthe oligonucleotides of the invention are prepared as SATE[(S-acetyl-2-thioethyl) phosphate] derivatives according to the methodsdisclosed in WO 93/24510 to Gosselin et al., published Dec. 9, 1993 orin WO 94/26764 and U.S. Pat. No. 5,770,713 to Imbach et al.

[0091] The term “pharmaceutically acceptable salts” refers tophysiologically and pharmaceutically acceptable salts of the compoundsof the invention: i.e., salts that retain the desired biologicalactivity of the parent compound and do not impart undesiredtoxicological effects thereto. For oligonucleotides, preferred examplesof pharmaceutically acceptable salts and their uses are furtherdescribed in U.S. Pat. No. 6,287,860, which is incorporated herein inits entirety.

[0092] The present invention also includes pharmaceutical compositionsand formulations which include the antisense compounds of the invention.The pharmaceutical compositions of the present invention may beadministered in a number of ways depending upon whether local orsystemic treatment is desired and upon the area to be treated.Administration may be topical (including ophthalmic and to mucousmembranes including vaginal and rectal delivery), pulmonary, e.g., byinhalation or insufflation of powders or aerosols, including bynebulizer; intratracheal, intranasal, epidermal and transdermal), oralor parenteral. Parenteral administration includes intravenous,intraarterial, subcutaneous, intraperitoneal or intramuscular injectionor infusion; or intracranial, e.g., intrathecal or intraventricular,administration. Oligonucleotides with at least one 2′-O-methoxyethylmodification are believed to be particularly useful for oraladministration. Pharmaceutical compositions and formulations for topicaladministration may include transdermal patches, ointments, lotions,creams, gels, drops, suppositories, sprays, liquids and powders.Conventional pharmaceutical carriers, aqueous, powder or oily bases,thickeners and the like may be necessary or desirable. Coated condoms,gloves and the like may also be useful.

[0093] The pharmaceutical formulations of the present invention, whichmay conveniently be presented in unit dosage form, may be preparedaccording to conventional techniques well known in the pharmaceuticalindustry. Such techniques include the step of bringing into associationthe active ingredients with the pharmaceutical carrier(s) orexcipient(s). In general, the formulations are prepared by uniformly andintimately bringing into association the active ingredients with liquidcarriers or finely divided solid carriers or both, and then, ifnecessary, shaping the product.

[0094] The compositions of the present invention may be formulated intoany of many possible dosage forms such as, but not limited to, tablets,capsules, gel capsules, liquid syrups, soft gels, suppositories, andenemas. The compositions of the present invention may also be formulatedas suspensions in aqueous, non-aqueous or mixed media. Aqueoussuspensions may further contain substances which increase the viscosityof the suspension including, for example, sodium carboxymethylcellulose,sorbitol and/or dextran. The suspension may also contain stabilizers.

[0095] Pharmaceutical compositions of the present invention include, butare not limited to, solutions, emulsions, foams and liposome-containingformulations. The pharmaceutical compositions and formulations of thepresent invention may comprise one or more penetration enhancers,carriers, excipients or other active or inactive ingredients.

[0096] Emulsions are typically heterogenous systems of one liquiddispersed in another in the form of droplets usually exceeding 0.1 μm indiameter. Emulsions may contain additional, components in addition tothe dispersed phases, and the active drug which may be present as asolution in either the aqueous phase, oily phase or itself as a separatephase. Microemulsions are included as an embodiment of the presentinvention. Emulsions and their uses are well known in the art and arefurther described in U.S. Pat. No. 6,287,860, which is incorporatedherein in its entirety.

[0097] Formulations of the present invention include liposomalformulations. As used in the present invention, the term “liposome”means a vesicle composed of amphiphilic lipids arranged in a sphericalbilayer or bilayers. Liposomes are unilamellar or multilamellar vesicleswhich have a membrane formed from a lipophilic material and an aqueousinterior that contains the composition to be delivered. Cationicliposomes are positively charged liposomes which are believed tointeract with negatively charged DNA molecules to form a stable complex.Liposomes that are pH-sensitive or negatively-charged are believed toentrap DNA rather than complex with it. Both cationic and noncationicliposomes have been used to deliver DNA to cells.

[0098] Liposomes also include “sterically stabilized” liposomes, a termwhich, as used herein, refers to liposomes comprising one or morespecialized lipids that, when incorporated into liposomes, result inenhanced circulation lifetimes relative to liposomes lacking suchspecialized lipids. Examples of sterically stabilized liposomes arethose in which part of the vesicle-forming lipid portion of the liposomecomprises one or more glycolipids or is derivatized with one or morehydrophilic polymers, such as a polyethylene glycol (PEG) moiety.Liposomes and their uses are further described in U.S. Pat. No.6,287,860, which is incorporated herein in its entirety.

[0099] The pharmaceutical formulations and compositions of the presentinvention may also include surfactants. The use of surfactants in drugproducts, formulations and in emulsions is well known in the art.Surfactants and their uses are further described in U.S. Pat. No.6,287,860, which is incorporated herein in its entirety.

[0100] In one embodiment, the present invention employs variouspenetration enhancers to effect the efficient delivery of nucleic acids,particularly oligonucleotides. In addition to aiding the diffusion ofnon-lipophilic drugs across cell membranes, penetration enhancers alsoenhance the permeability of lipophilic drugs. Penetration enhancers maybe classified as belonging to one of five broad categories, i.e.,surfactants, fatty acids, bile salts, chelating agents, andnon-chelating non-surfactants. Penetration enhancers and their uses arefurther described in U.S. Pat. No. 6,287,860, which is incorporatedherein in its entirety.

[0101] One of skill in the art will recognize that formulations areroutinely designed according to their intended use, i.e. route ofadministration.

[0102] Preferred formulations for topical administration include thosein which the oligonucleotides of the invention are in admixture with atopical delivery agent such as lipids, liposomes, fatty acids, fattyacid esters, steroids, chelating agents and surfactants. Preferredlipids and liposomes include neutral (e.g. dioleoylphosphatidyl DOPEethanolamine, dimyristoylphosphatidyl choline DMPC,distearolyphosphatidyl choline) negative (e.g. dimyristoylphosphatidylglycerol DMPG) and cationic (e.g. dioleoyltetramethylaminopropyl DOTAPand dioleoylphosphatidyl ethanolamine DOTMA).

[0103] For topical or other administration, oligonucleotides of theinvention may be encapsulated within liposomes or may form complexesthereto, in particular to cationic liposomes. Alternatively,oligonucleotides may be complexed to lipids, in particular to cationiclipids. Preferred fatty acids and esters, pharmaceutically acceptablesalts thereof, and their uses are further described in U.S. Pat. No.6,287,860, which is incorporated herein in its entirety. Topicalformulations are described in detail in U.S. patent application Ser. No.09/315,298 filed on May 20, 1999, which is incorporated herein byreference in its entirety.

[0104] Compositions and formulations for oral administration includepowders or granules, microparticulates, nanoparticulates, suspensions orsolutions in water or non-aqueous media, capsules, gel capsules,sachets, tablets or minitablets. Thickeners, flavoring agents, diluents,emulsifiers, dispersing aids or binders may be desirable. Preferred oralformulations are those in which oligonucleotides of the invention areadministered in conjunction with one or more penetration enhancerssurfactants and chelators. Preferred surfactants include fatty acidsand/or esters or salts thereof, bile acids and/or salts thereof.Preferred bile acids/salts and fatty acids and their uses are furtherdescribed in U.S. Pat. No. 6,287,860, which is incorporated herein inits entirety. Also preferred are combinations of penetration enhancers,for example, fatty acids/salts in combination with bile acids/salts. Aparticularly preferred combination is the sodium salt of lauric acid,capric acid and UDCA. Further penetration enhancers includepolyoxyethylene-9-lauryl ether, polyoxyethylene-20-cetyl etheroligonucleotides of the invention may be delivered orally, in granularform including sprayed dried particles, or complexed to form micro ornanoparticles. Oligonucleotide complexing agents and their uses arefurther described in U.S. Pat. No. 6,287,860, which is incorporatedherein in its entirety. Oral formulations for oligonucleotides and theirpreparation are described in detail in U.S. application Ser. No.09/108,673 (filed Jul. 1, 1998), Ser. No. 09/315,298 (filed May 20,1999) and Ser. No. 10/071,822, filed Feb. 8, 2002, each of which isincorporated herein by reference in their entirety.

[0105] Compositions and formulations for parenteral, intrathecal orintraventricular administration may include sterile aqueous solutionswhich may also contain buffers, diluents and other suitable additivessuch as, but not limited to, penetration enhancers, carrier compoundsand other pharmaceutically acceptable carriers or excipients.

[0106] Certain embodiments of the invention provide pharmaceuticalcompositions containing one or more oligomeric compounds and one or moreother chemotherapeutic agents which function by a non-antisensemechanism. Examples of such chemotherapeutic agents include but are notlimited to cancer chemotherapeutic drugs such as daunorubicin,daunomycin, dactinomycin, doxorubicin, epirubicin, idarubicin,esorubicin, bleomycin, mafosfamide, ifosfamide, cytosine arabinoside,bis-chloroethylnitrosurea, busulfan, mitomycin C, actinomycin D,mithramycin, prednisone, hydroxyprogesterone, testosterone, tamoxifen,dacarbazine, procarbazine, hexamethylmelamine, pentamethylmelamine,mitoxantrone, amsacrine, chlorambucil, methylcyclohexylnitrosurea,nitrogen mustards, melphalan, cyclophosphamide, 6-mercaptopurine,6-thioguanine, cytarabine, 5-azacytidine, hydroxyurea, deoxyco-formycin,4-hydroxyperoxycyclophosphoramide, 5-fluorouracil (5-FU),5-fluorodeoxyuridine (5-FUdR), methotrexate (MTX), colchicine, taxol,vincristine, vinblastine, etoposide (VP-16), trimetrexate, irinotecan,topotecan, gemcitabine, teniposide, cisplatin and diethylstilbestrol(DES). When used with the compounds of the invention, suchchemotherapeutic agents may be used individually (e.g., 5-FU andoligonucleotide), sequentially (e.g., 5-FU and oligonucleotide for aperiod of time followed by MTX and oligonucleotide), or in combinationwith one or more other such chemotherapeutic agents (e.g., 5-FU, MTX andoligonucleotide, or 5-FU, radiotherapy and oligonucleotide).Anti-inflammatory drugs, including but not limited to nonsteroidalanti-inflammatory drugs and corticosteroids, and antiviral drugs,including but not limited to ribivirin, vidarabine, acyclovir andganciclovir, may also be combined in compositions of the invention.Combinations of antisense compounds and other non-antisense drugs arealso within the scope of this invention. Two or more combined compoundsmay be used together or sequentially.

[0107] In another related embodiment, compositions of the invention maycontain one or more antisense compounds, particularly oligonucleotides,targeted to a first nucleic acid and one or more additional antisensecompounds targeted to a second nucleic acid target. Alternatively,compositions of the invention may contain two or more antisensecompounds targeted to different regions of the same nucleic acid target.Numerous examples of antisense compounds are known in the art. Two ormore combined compounds may be used together or sequentially.

[0108] H. Dosing

[0109] The formulation of therapeutic compositions and their subsequentadministration (dosing) is believed to be within the skill of those inthe art. Dosing is dependent on severity and responsiveness of thedisease state to be treated, with the course of treatment lasting fromseveral days to several months, or until a cure is effected or adiminution of the disease state is achieved. Optimal dosing schedulescan be calculated from measurements of drug accumulation in the body ofthe patient. Persons of ordinary skill can easily determine optimumdosages, dosing methodologies and repetition rates. Optimum dosages mayvary depending on the relative potency of individual oligonucleotides,and can generally be estimated based on EC₅₀S found to be effective inin vitro and in vivo animal models. In general, dosage is from 0.01 ugto 100 g per kg of body weight, and may be given once or more daily,weekly, monthly or yearly, or even once every 2 to 20 years. Persons ofordinary skill in the art can easily estimate repetition rates fordosing based on measured residence times and concentrations of the drugin bodily fluids or tissues. Following successful treatment, it may bedesirable to have the patient undergo maintenance therapy to prevent therecurrence of the disease state, wherein the oligonucleotide isadministered in maintenance doses, ranging from 0.01 ug to 100 g per kgof body weight, once or more daily, to once every 20 years.

[0110] While the present invention has been described with specificityin accordance with certain of its preferred embodiments, the followingexamples serve only to illustrate the invention and are not intended tolimit the same.

EXAMPLES Example 1

[0111] Synthesis of Nucleoside Phosphoramidites

[0112] The following compounds, including amidites and theirintermediates were prepared as described in U.S. Pat. No. 6,426,220 andpublished PCT WO 02/36743; 5′-O-Dimethoxytrityl-thymidine intermediatefor 5-methyl dC amidite, 5′-O-Dimethoxytrityl-2′-deoxy-5-methylcytidineintermediate for 5-methyl-dC amidite,5′-O-Dimethoxytrityl-2′-deoxy-N-4-benzoyl-5-methylcytidine penultimateintermediate for 5-methyl dC amidite,[5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-deoxy-N⁴-benzoyl-5-methylcytidin-3′-O-yl]-2-cyanoethyl-N,N-diisopropylphosphoramidite(5-methyl dC amidite), 2′-Fluorodeoxyadenosine, 2′-Fluorodeoxyguanosine,2′-Fluorouridine, 2′-Fluorodeoxycytidine, 2′-O-(2-Methoxyethyl) modifiedamidites, 2′-O-(2-methoxyethyl)-5-methyluridine intermediate,5′-O-DMT-2′-O-(2-methoxyethyl)-5-methyluridine penultimate intermediate,[5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-O-(2-methoxyethyl)-5-methyluridin-3′-O-yl]-2-cyanoethyl-N,N-diisopropylphosphoramidite(MOE T amidite),5′-O-Dimethoxytrityl-2′-O-(2-methoxyethyl)-5-methylcytidineintermediate,5′-O-dimethoxytrityl-2′-O-(2-methoxyethyl)-N⁴-benzoyl-5-methyl-cytidinepenultimate intermediate,[5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-O-(2-methoxyethyl)-N⁶-benzoyl-5-methylcytidin-3′-O-yl]-2-cyanoethyl-N,N-diisopropylphosphoramidite(MOE 5-Me-C amidite),[5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-O-(2-methoxyethyl)-N⁶-benzoyladenosin-3′-O-yl]-2-cyanoethyl-N,N-diisopropylphosphoramidite(MOE A amdite),[5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-O-(2-methoxyethyl)-N⁴-isobutyrylguanosin-3′-O-yl]-2-cyanoethyl-N,N-diisopropylphosphoramidite(MOE G amidite), 2′-O-(Aminooxyethyl) nucleoside amidites and2′-O-(dimethylamino-oxyethyl) nucleoside amidites,2′-(Dimethylaminooxyethoxy) nucleoside amidites,5′-O-tert-Butyldiphenylsilyl-O²-2′-anhydro-5-methyluridine,5′-O-tert-Butyldiphenylsilyl-2′-O-(2-hydroxyethyl)-5-methyluridine,2′-O-([2-phthalimidoxy)ethyl]-5′-t-butyldiphenylsilyl-5-methyluridine,5′-O-tert-butyldiphenylsilyl-2′-O-[(2-formadoximinooxy)ethyl]-5-methyluridine,5′-O-LertButyldiphenylsilyl-2′-O-[N,Ndimethylaminooxyethyl]-5-methyluridine,2′-O-(dimethylaminooxyethyl)-5-methyluridine,5′-O-DMT-2′-O-(dimethylaminooxyethyl)-5-methyluridine,5′-O-DMT-2′-O-(2-N,N-dimethylaminooxyethyl)-5-methyluridine-3′-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite],2′-(Aminooxyethoxy) nucleoside amidites,N2-isobutyryl-6-O-diphenylcarbamoyl-2′-O-(2-ethylacetyl)-5′-O-(4,4′-dimethoxytrityl)guanosine-3′-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite],2′-dimethylaminoethoxyethoxy (2′-DMAEOE) nucleoside amidites,2′-O-[2(2-N,N-dimethylaminoethoxy)ethyl]-5-methyl uridine,5′-O-dimethoxytrityl-2′-O-[2(2-N,N-dimethylaminoethoxy)-ethyl)]-5-methyluridine and5′-O-Dimethoxytrityl-2′-O-[2(2-N,N-dimethylaminoethoxy)-ethyl)]-5-methyluridine-3′-O-(cyanoethyl-N,N-diisopropyl)phosphoramidite.

Example 2

[0113] Oligonucleotide and Oligonucleoside Synthesis

[0114] The antisense compounds used in accordance with this inventionmay be conveniently and routinely made through the well-known techniqueof solid phase synthesis. Equipment for such synthesis is sold byseveral vendors including, for example, Applied Biosystems (Foster City,Calif.). Any other means for such synthesis known in the art mayadditionally or alternatively be employed. It is well known to usesimilar techniques to prepare oligonucleotides such as thephosphorothioates and alkylated derivatives.

[0115] Oligonucleotides: Unsubstituted and substituted phosphodiester(P═O) oligonucleotides are synthesized on an automated DNA synthesizer(Applied Biosystems model 394) using standard phosphoramidite chemistrywith oxidation by iodine.

[0116] Phosphorothioates (P═S) are synthesized similar to phosphodiesteroligonucleotides with the following exceptions: thiation was effected byutilizing a 10% w/v solution of, 3,H-1,2-benzodithiole-3-one 1,1-dioxidein acetonitrile for the oxidation of the phosphite linkages. Thethiation reaction step time was increased to 180 sec and preceded by thenormal capping step. After cleavage from the CPG column and deblockingin concentrated ammonium hydroxide at 55° C. (12-16 hr), theoligonucleotides were recovered by precipitating with >3 volumes ofethanol from a 1 M NH₄OAc solution. Phosphinate oligonucleotides areprepared as described in U.S. Pat. No. 5,508,270, herein incorporated byreference.

[0117] Alkyl phosphonate oligonucleotides are prepared as described inU.S. Pat. No. 4,469,863, herein incorporated by reference.

[0118] 3′-Deoxy-3′-methylene phosphonate oligonucleotides are preparedas described in U.S. Pat. Nos. 5,610,289 or 5,625,050, hereinincorporated by reference.

[0119] Phosphoramidite oligonucleotides are prepared as described inU.S. Pat. No. 5,256,775 or U.S. Pat. No. 5,366,878, herein incorporatedby reference.

[0120] Alkylphosphonothioate oligonucleotides are prepared as describedin published PCT applications PCT/US94/00902 and PCT/US93/06976(published as WO 94/17093 and WO 94/02499, respectively), hereinincorporated by reference.

[0121] 3′-Deoxy-3′-amino phosphoramidate oligonucleotides are preparedas described in U.S. Pat. No. 5,476,925, herein incorporated byreference.

[0122] Phosphotriester oligonucleotides are prepared as described inU.S. Pat. No. 5,023,243, herein incorporated by reference.

[0123] Borano phosphate oligonucleotides are prepared as described inU.S. Pat. Nos. 5,130,302 and 5,177,198, both herein incorporated byreference.

[0124] Oligonucleosides: Methylenemethylimino linked oligonucleosides,also identified as MMI linked oligonucleosides, methylenedimethylhydrazolinked oligonucleosides, also identified as MDH linked oligonucleosides,and methylenecarbonylamino linked oligonucleosides, also identified asamide-3 linked oligonucleosides, and methyleneaminocarbonyl linkedoligonucleosides, also identified as amide-4 linked oligonucleosides, aswell as mixed backbone compounds having, for instance, alternating MMIand P═O or P═S linkages are prepared as described in U.S. Pat. Nos.5,378,825, 5,386,023, 5,489,677, 5,602,240 and 5,610,289, all of whichare herein incorporated by reference.

[0125] Formacetal and thioformacetal linked oligonucleosides areprepared as described in U.S. Pat. Nos. 5,264,562 and 5,264,564, hereinincorporated by reference.

[0126] Ethylene oxide linked oligonucleosides are prepared as describedin U.S. Pat. No. 5,223,618, herein incorporated by reference.

Example 3

[0127] RNA Synthesis

[0128] In general, RNA synthesis chemistry is based on the selectiveincorporation of various protecting groups at strategic intermediaryreactions. Although one of ordinary skill in the art will understand theuse of protecting groups in organic synthesis, a useful class ofprotecting groups includes silyl ethers. In particular bulky silylethers are used to protect the 5′-hydroxyl in combination with anacid-labile orthoester protecting group on the 2′-hydroxyl. This set ofprotecting groups is then used with standard solid-phase synthesistechnology. It is important to lastly remove the acid labile orthoesterprotecting group after all other synthetic steps. Moreover, the earlyuse of the silyl protecting groups during synthesis ensures facileremoval when desired, without undesired deprotection of 2′ hydroxyl.

[0129] Following this procedure for the sequential protection of the5′-hydroxyl in combination with protection of the 2′-hydroxyl byprotecting groups that are differentially removed and are differentiallychemically labile, RNA oligonucleotides were synthesized.

[0130] RNA oligonucleotides are synthesized in a stepwise fashion. Eachnucleotide is added sequentially (3′- to 5′-direction) to a solidsupport-bound oligonucleotide. The first nucleoside at the 3′-end of thechain is covalently attached to a solid support. The nucleotideprecursor, a ribonucleoside phosphoramidite, and activator are added,coupling the second base onto the 5′-end of the first nucleoside. Thesupport is washed and any unreacted 5′-hydroxyl groups are capped withacetic anhydride to yield 5′-acetyl moieties. The linkage is thenoxidized to the more stable and ultimately desired P(V) linkage. At theend of the nucleotide addition cycle, the 5′-silyl group is cleaved withfluoride. The cycle is repeated for each subsequent nucleotide.

[0131] Following synthesis, the methyl protecting groups on thephosphates are cleaved in 30 minutes utilizing 1 Mdisodium-2-carbamoyl-2-cyanoethylene-1,1-dithiolate trihydrate (S₂Na₂)in DMF. The deprotection solution is washed from the solid support-boundoligonucleotide using water. The support is then treated with 40%methylamine in water for 10 minutes at 55° C. This releases the RNAoligonucleotides into solution, deprotects the exocyclic amines, andmodifies the 2′-groups. The oligonucleotides can be analyzed by anionexchange HPLC at this stage.

[0132] The 2′-orthoester groups are the last protecting groups to beremoved. The ethylene glycol monoacetate orthoester protecting groupdeveloped by Dharmacon Research, Inc. (Lafayette, Colo.), is one exampleof a useful orthoester protecting group which, has the followingimportant properties. It is stable to the conditions of nucleosidephosphoramidite synthesis and oligonucleotide synthesis. However, afteroligonucleotide synthesis the oligonucleotide is treated withmethylamine which not only cleaves the oligonucleotide from the solidsupport but also removes the acetyl groups from the orthoesters. Theresulting 2-ethyl-hydroxyl substituents on the orthoester are lesselectron withdrawing than the acetylated precursor. As a result, themodified orthoester becomes more labile to acid-catalyzed hydrolysis.Specifically, the rate of cleavage is approximately 10 times fasterafter the acetyl groups are removed. Therefore, this orthoesterpossesses sufficient stability in order to be compatible witholigonucleotide synthesis and yet, when subsequently modified, permitsdeprotection to be carried out under relatively mild aqueous conditionscompatible with the final RNA oligonucleotide product.

[0133] Additionally, methods of RNA synthesis are well known in the art(Scaringe, S. A. Ph.D. Thesis, University of Colorado, 1996; Scaringe,S. A., et al., J. Am. Chem. Soc., 1998, 120, 11820-11821; Matteucci, M.D. and Caruthers, M. H. J. Am. Chem. Soc., 1981, 103, 3185-3191;Beaucage, S. L. and Caruthers, M. H. Tetrahedron Lett., 1981, 22,1859-1862; Dahl, B. J., et al., Acta Chem. Scand,. 1990, 44, 639-641;Reddy, M. P., et al., Tetrahedrom Lett., 1994, 25, 4311-4314; Wincott,F. et al., Nucleic Acids Res., 1995, 23, 2677-2684; Griffin, B. E., etal., Tetrahedron, 1967, 23, 2301-2313; Griffin, B. E., et al.,Tetrahedron, 1967, 23, 2315-2331).

[0134] RNA antisense compounds (RNA oligonucleotides) of the presentinvention can be synthesized by the methods herein or purchased fromDharmacon Research, Inc (Lafayette, Colo.). Once synthesized,complementary RNA antisense compounds can then be annealed by methodsknown in the art to form double stranded (duplexed) antisense compounds.For example, duplexes can be formed by combining 30 μl of each of thecomplementary strands of RNA oligonucleotides (50 uM RNA oligonucleotidesolution) and 15 μl of 5×annealing buffer (100 mm potassium acetate, 30mM HEPES-KOH pH 7.4, 2 mM magnesium acetate) followed by heating for 1minute at 90° C., then 1 hour at 37° C. The resulting duplexed antisensecompounds can be used in kits, assays, screens, or other methods toinvestigate the role of a target nucleic acid.

Example 4

[0135] Synthesis of Chimeric Oligonucleotides

[0136] Chimeric oligonucleotides, oligonucleosides or mixedoligonucleotides/oligonucleosides of the invention can be of severaldifferent types. These include a first type wherein the “gap” segment oflinked nucleosides is positioned between 5′ and 3′ “wing” segments oflinked nucleosides and a second “open end” type wherein the “gap”segment is located at either the 3′ or the 5′ terminus of the oligomericcompound. Oligonucleotides of the first type are also known in the artas “gapmers” or gapped oligonucleotides. Oligonucleotides of the secondtype are also known in the art as “hemimers” or “wingmers”.

[0137] [2′-O-Me]--[2′-deoxy]--[2′-O-Me] Chimeric Phosphorothioateoligonucleotides

[0138] Chimeric oligonucleotides having 2′-O-alkyl phosphorothioate and2′-deoxy phosphorothioate oligonucleotide segments are synthesized usingan Applied Biosystems automated DNA synthesizer Model 394, as above.Oligonucleotides are synthesized using the automated synthesizer and2′-deoxy-5′-dimethoxytrityl-3′-O-phosphoramidite for the DNA portion and5′-dimethoxytrityl-2′-O-methyl-3′-O-phosphoramidite for 5′ and 3′ wings.The standard synthesis cycle is modified by incorporating coupling stepswith increased reaction times for the5′-dimethoxytrityl-2′-O-methyl-3′-O-phosphoramidite. The fully protectedoligonucleotide is cleaved from the support and deprotected inconcentrated ammonia (NH₄OH) for 12-16 hr at 55° C. The deprotectedoligo is then recovered by an appropriate method (precipitation, columnchromatography, volume reduced in vacuo and analyzedspetrophotometrically for yield and for purity by capillaryelectrophoresis and by mass spectrometry.

[0139][2′-O-(2-Methoxyethyl)]--[2′-deoxy]--[2′-O-(Methoxyethyl)]ChimericPhosphorothioate Oligonucleotides

[0140] [2′-O-(2-methoxyethyl)]--[2′-deoxy]--[-2′-O-(methoxyethyl)]chimeric phosphorothioate oligonucleotides were prepared as per theprocedure above for the 2′-O-methyl chimeric oligonucleotide, with thesubstitution of 2′-O(methoxyethyl) amidites for the 2′-O-methylamidites.

[0141] [2′-O-(2-Methoxyethyl)Phosphodiester]--[2′-deoxyPhosphorothioate]--[2′-O-(2-Methoxyethyl) Phosphodiester]ChimericOligonucleotides

[0142] [2′-O-(2-methoxyethyl phosphodiester]--[2′-deoxyphosphorothioate]--[2′-O-(methoxyethyl) phosphodiester] chimericoligonucleotides are prepared as per the above procedure for the2′-O-methyl chimeric oligonucleotide with the substitution of2′-O-(methoxyethyl) amidites for the 2′O-methyl amidites, oxidation withiodine to generate the phosphodiester internucleotide linkages withinthe wing portions of the chimeric structures and sulfurization utilizing3,H-1,2 benzodithiole-3-one 1,1 dioxide (Beaucage Reagent) to generatethe phosphorothioate internucleotide linkages for the center gap.

[0143] Other chimeric oligonucleotides, chimeric oligonucleosides andmixed chimeric oligonucleotides/oligonucleosides are synthesizedaccording to U.S. Pat. No. 5,623,065, herein incorporated by reference.

Example 5

[0144] Design and Screening of Duplexed Antisense Compounds TargetingInterleukin 22 Receptor

[0145] In accordance with the present invention, a series of nucleicacid duplexes comprising the antisense compounds of the presentinvention and their complements can be designed to target interleukin 22receptor. The nucleobase sequence of the antisense strand of the duplexcomprises at least a portion of an oligonucleotide in Table 1. The endsof the strands may be modified by the addition of one or more natural ormodified nucleobases to form an overhang. The sense strand of the dsRNAis then designed and synthesized as the complement of the antisensestrand and may also contain modifications or additions to eitherterminus. For example, in one embodiment, both strands of the dsRNAduplex would be complementary over the central nucleobases, each havingoverhangs at one or both termini.

[0146] For example, a duplex comprising an antisense strand having thesequence CGAGAGGCGGACGGGACCG and having a two-nucleobase overhang ofdeoxythymidine(dT) would have the following structure:  cgagaggcggacgggaccgTT Antisense Strand   |||||||||||||||||||TTgctctccgcctgccctggc Complement

[0147] RNA strands of the duplex can be synthesized by methods disclosedherein or purchased from Dharmacon Research Inc., (Lafayette, Colo.).Once synthesized, the complementary strands are annealed. The singlestrands are aliquoted and diluted to a concentration of 50 uM. Oncediluted, 30 uL of each strand is combined with 15 uL of a 5×solution ofannealing buffer. The final concentration of said buffer is 100 mMpotassium acetate, 30 mM HEPES-KOH pH 7.4, and 2 mM magnesium acetate.The final volume is 75 uL. This solution is incubated for 1 minute at90° C. and then centrifuged for 15 seconds. The tube is allowed to sitfor 1 hour at 37° C. at which time the dsRNA duplexes are used inexperimentation. The final concentration of the dsRNA duplex is 20 uM.This solution can be stored frozen (−20° C.) and freeze-thawed up to 5times.

[0148] Once prepared, the duplexed antisense compounds are evaluated fortheir ability to modulate interleukin 22 receptor expression.

[0149] When cells reached 80% confluency, they are treated with duplexedantisense compounds of the invention. For cells grown in 96-well plates,wells are washed once with 200 μL OPTI-MEM-1 reduced-serum medium (GibcoBRL) and then treated with 130 μL of OPTI-MEM-1 containing 12 μg/mLLIPOFECTIN (Gibco BRL) and the desired duplex antisense compound at afinal concentration of 200 nM. After 5 hours of treatment, the medium isreplaced with fresh medium. Cells are harvested 16 hours aftertreatment, at which time RNA is isolated and target reduction measuredby RT-PCR.

Example 6

[0150] Oligonucleotide Isolation

[0151] After cleavage from the controlled pore glass solid support anddeblocking in concentrated ammonium hydroxide at 55° C. for 12-16 hours,the oligonucleotides or oligonucleosides are recovered by precipitationout of 1 M NH₄OAc with >3 volumes of ethanol. Synthesizedoligonucleotides were analyzed by electrospray mass spectroscopy(molecular weight determination) and by capillary gel electrophoresisand judged to be at least 70% full length material. The relative amountsof phosphorothioate and phosphodiester linkages obtained in thesynthesis was determined by the ratio of correct molecular weightrelative to the −16 amu product (+/−32+/−48). For some studiesoligonucleotides were purified by HPLC, as described by Chiang et al.,J. Biol. Chem. 1991, 266, 18162-18171. Results obtained withHPLC-purified material were similar to those obtained with non-HPLCpurified material.

Example 7

[0152] Oligonucleotide Synthesis—96 Well Plate Format

[0153] Oligonucleotides were synthesized via solid phase P(III)phosphoramidite chemistry on an automated synthesizer capable ofassembling 96 sequences simultaneously in a 96-well format.Phosphodiester internucleotide linkages were afforded by oxidation withaqueous iodine. Phosphorothioate internucleotide linkages were generatedby sulfurization utilizing 3,H-1,2 benzodithiole-3-one 1,1 dioxide(Beaucage Reagent) in anhydrous acetonitrile. Standard base-protectedbeta-cyanoethyl-diiso-propyl phosphoramidites were purchased fromcommercial vendors (e.g. PE-Applied Biosystems, Foster City, Calif., orPharmacia, Piscataway, N.J.). Non-standard nucleosides are synthesizedas per standard or patented methods. They are utilized as base protectedbeta-cyanoethyldiisopropyl phosphoramidites.

[0154] Oligonucleotides were cleaved from support and deprotected withconcentrated NH₄OH at elevated temperature (55-60° C.) for 12-16 hoursand the released product then dried in vacuo. The dried product was thenre-suspended in sterile water to afford a master plate from which allanalytical and test plate samples are then diluted utilizing roboticpipettors.

Example 8

[0155] Oligonucleotide Analysis—96-Well Plate Format

[0156] The concentration of oligonucleotide in each well was assessed bydilution of samples and UV absorption spectroscopy. The full-lengthintegrity of the individual products was evaluated by capillaryelectrophoresis (CE) in either the 96-well format (Beckman P/ACETM MDQ)or, for individually prepared samples, on a commercial CE apparatus(e.g., Beckman P/ACE™ 5000, ABI 270). Base and backbone composition wasconfirmed by mass analysis of the compounds utilizing electrospray-massspectroscopy. All assay test plates were diluted from the master plateusing single and multi-channel robotic pipettors. Plates were judged tobe acceptable if at least 85% of the compounds on the plate were atleast 85% full length.

Example 9

[0157] Cell Culture and Oligonucleotide Treatment

[0158] The effect of antisense compounds on target nucleic acidexpression can be tested in any of a variety of cell types provided thatthe target nucleic acid is present at measurable levels. This can beroutinely determined using, for example, PCR or Northern blot analysis.The following cell types are provided for illustrative purposes, butother cell types can be routinely used, provided that the target isexpressed in the cell type chosen. This can be readily determined bymethods routine in the art, for example Northern blot analysis,ribonuclease protection assays, or RT-PCR.

[0159] T-24 Cells:

[0160] The human transitional cell bladder carcinoma cell line T-24 wasobtained from the American Type Culture Collection (ATCC) (Manassas,Va.). T-24 cells were routinely cultured in complete McCoy's 5A basalmedia (Invitrogen Corporation, Carlsbad, Calif.) supplemented with 10%fetal calf serum (Invitrogen Corporation, Carlsbad, Calif.), penicillin100 units per mL, and streptomycin 100 micrograms per mL (InvitrogenCorporation, Carlsbad, Calif.). Cells were routinely passaged bytrypsinization and dilution when they reached 90% confluence. Cells wereseeded into 96-well plates (Falcon-Primaria #353872) at a density of7000 cells/well for use in RT-PCR analysis.

[0161] For Northern blotting or other analysis, cells may be seeded onto100 mm or other standard tissue culture plates and treated similarly,using appropriate volumes of medium and oligonucleotide.

[0162] A549 Cells:

[0163] The human lung carcinoma cell line A549 was obtained from theAmerican Type Culture Collection (ATCC) (Manassas, Va.). A549 cells wereroutinely cultured in DMEM basal media (Invitrogen Corporation,Carlsbad, Calif.) supplemented with 10% fetal calf serum (InvitrogenCorporation, Carlsbad, Calif.), penicillin 100 units per mL, andstreptomycin 100 micrograms per mL (Invitrogen Corporation, Carlsbad,Calif.). Cells were routinely passaged by trypsinization and dilutionwhen they reached 90% confluence.

[0164] NHDF Cells:

[0165] Human neonatal dermal fibroblast (NHDF) were obtained from theClonetics Corporation (Walkersville, Md.). NHDFs were routinelymaintained in Fibroblast Growth Medium (Clonetics Corporation,Walkersville, Md.) supplemented as recommended by the supplier. Cellswere maintained for up to 10 passages as recommended by the supplier.

[0166] HEK Cells:

[0167] Human embryonic keratinocytes (HEK) were obtained from theClonetics Corporation (Walkersville, Md.). HEKs were routinelymaintained in Keratinocyte Growth Medium (Clonetics Corporation,Walkersville, Md.) formulated as recommended by the supplier. Cells wereroutinely maintained for up to 10 passages as recommended by thesupplier.

[0168] HepG2 Cells:

[0169] The human hepatoblastoma cell line HepG2 was obtained from theAmerican Type Culture Collection (Manassas, Va.). HepG2 cells wereroutinely cultured in Eagle's MEM supplemented with 10% fetal calfserum, non-essential amino acids, and 1 mM sodium pyruvate (Gibco/LifeTechnologies, Gaithersburg, Md.). Cells were routinely passaged bytrypsinization and dilution when they reached 90% confluence. Cells wereseeded into 96-well plates (Falcon-Primaria #3872) at a density of 7000cells/well for use in RT-PCR analysis.

[0170] For Northern blotting or other analyses, cells may be seeded onto100 mm or other standard tissue culture plates and treated similarly,using appropriate volumes of medium and oligonucleotide.

[0171] Treatment with Antisense Compounds:

[0172] When cells reached 65-75% confluency, they were treated witholigonucleotide. For cells grown in 96-well plates, wells were washedonce with 100 μL OPTI-MEM™-1 reduced-serum medium (InvitrogenCorporation, Carlsbad, Calif.) and then treated with 130 μL ofOPTI-MEM™-1 containing 3.75 μg/mL LIPOFECTIN™ (Invitrogen Corporation,Carlsbad, Calif.) and the desired concentration of oligonucleotide.Cells are treated and data are obtained in triplicate. After 4-7 hoursof treatment at 37° C., the medium was replaced with fresh medium. Cellswere harvested 16-24 hours after oligonucleotide treatment.

[0173] The concentration of oligonucleotide used varies from cell lineto cell line. To determine the optimal oligonucleotide concentration fora particular cell line, the cells are treated with a positive controloligonucleotide at a range of concentrations. For human cells thepositive control oligonucleotide is selected from either ISIS 13920(TCCGTCATCGCTCCTCAGGG, SEQ ID NO: 1) which is targeted to human H-ras,or ISIS 18078, (GTGCGCGCGAGCCCGAAATC, SEQ ID NO: 2) which is targeted tohuman Jun-N-terminal kinase-2 (JNK2). Both controls are2′-O-methoxyethyl gapmers (2′-O-methoxyethyls shown in bold) with aphosphorothioate backbone. For mouse or rat cells the positive controloligonucleotide is ISIS 15770, ATGCATTCTGCCCCCAAGGA, SEQ ID NO: 3, a2′-O-methoxyethyl gapmer (2′-O-methoxyethyls shown in bold) with aphosphorothioate backbone which is targeted to both mouse and rat c-raf.The concentration of positive control oligonucleotide that results in80% inhibition of c-H-ras (for ISIS 13920), JNK2 (for ISIS 18078) orc-raf (for ISIS 15770) mRNA is then utilized as the screeningconcentration for new oligonucleotides in subsequent experiments forthat cell line. If 80% inhibition is not achieved, the lowestconcentration of positive control oligonucleotide that results in 60%inhibition of c-H-ras, JNK2 or c-raf mRNA is then utilized as theoligonucleotide screening concentration in subsequent experiments forthat cell line. If 60% inhibition is not achieved, that particular cellline is deemed as unsuitable for oligonucleotide transfectionexperiments. The concentrations of antisense oligonucleotides usedherein are from 50 nM to 300 nM.

Example 10

[0174] Analysis of Oligonucleotide Inhibition of Interleukin 22 ReceptorExpression

[0175] Antisense modulation of interleukin 22 receptor expression can beassayed in a variety of ways known in the art. For example, interleukin22 receptor mRNA levels can be quantitated by, e.g., Northern blotanalysis, competitive polymerase chain reaction (PCR), or real-time PCR(RT-PCR). Real-time quantitative PCR is presently preferred. RNAanalysis can be performed on total cellular RNA or poly(A)+ mRNA. Thepreferred method of RNA analysis of the present invention is the use oftotal cellular RNA as described in other examples herein. Methods of RNAisolation are well known in the art. Northern blot analysis is alsoroutine in the art. Real-time quantitative (PCR) can be convenientlyaccomplished using the commercially available ABI PRISM™ 7600, 7700, or7900 Sequence Detection System, available from PE-Applied Biosystems,Foster City, Calif. and used according to manufacturer's instructions.

[0176] Protein levels of interleukin 22 receptor can be quantitated in avariety of ways well known in the art, such as immunoprecipitation,Western blot analysis (immunoblotting), enzyme-linked immunosorbentassay (ELISA) or fluorescence-activated cell sorting (FACS). Antibodiesdirected to interleukin 22 receptor can be identified and obtained froma variety of sources, such as the MSRS catalog of antibodies (AerieCorporation, Birmingham, Mich.), or can be prepared via conventionalmonoclonal or polyclonal antibody generation methods well known in theart.

Example 11

[0177] Design of Phenotypic Assays and In Vivo Studies for the use ofInterleukin 22 Receptor Inhibitors

[0178] Phenotypic Assays

[0179] Once interleukin 22 receptor inhibitors have been identified bythe methods disclosed herein, the compounds are further investigated inone or more phenotypic assays, each having measurable endpointspredictive of efficacy in the treatment of a particular disease state orcondition. Phenotypic assays, kits and reagents for their use are wellknown to those skilled in the art and are herein used to investigate therole and/or association of interleukin 22 receptor in health anddisease. Representative phenotypic assays, which can be purchased fromany one of several commercial vendors, include those for determiningcell viability, cytotoxicity, proliferation or cell survival (MolecularProbes, Eugene, Oreg.; PerkinElmer, Boston, Mass.), protein-based assaysincluding enzymatic assays (Panvera, LLC, Madison, Wis.; BD Biosciences,Franklin Lakes, N.J.; Oncogene Research Products, San Diego, Calif.),cell regulation, signal transduction, inflammation, oxidative processesand apoptosis (Assay Designs Inc., Ann Arbor, Mich.), triglycerideaccumulation (Sigma-Aldrich, St. Louis, Mo.), angiogenesis assays, tubeformation assays, cytokine and hormone assays and metabolic assays(Chemicon International Inc., Temecula, Calif.; Amersham Biosciences,Piscataway, N.J.).

[0180] In one non-limiting example, cells determined to be appropriatefor a particular phenotypic assay (i.e., MCF-7 cells selected for breastcancer studies; adipocytes for obesity studies) are treated withinterleukin 22 receptor inhibitors identified from the in vitro studiesas well as control compounds at optimal concentrations which aredetermined by the methods described above. At the end of the treatmentperiod, treated and untreated cells are analyzed by one or more methodsspecific for the assay to determine phenotypic outcomes and endpoints.

[0181] Phenotypic endpoints include changes in cell morphology over timeor treatment dose as well as changes in levels of cellular componentssuch as proteins, lipids, nucleic acids, hormones, saccharides ormetals. Measurements of cellular status which include pH, stage of thecell cycle, intake or excretion of biological indicators by the cell,are also endpoints of interest.

[0182] Analysis of the geneotype of the cell (measurement of theexpression of one or more of the genes of the cell) after treatment isalso used as an indicator of the efficacy or potency of the interleukin22 receptor inhibitors. Hallmark genes, or those genes suspected to beassociated with a specific disease state, condition, or phenotype, aremeasured in both treated and untreated cells.

[0183] In Vivo Studies

[0184] The individual subjects of the in vivo studies described hereinare warm-blooded vertebrate animals, which includes humans.

[0185] The clinical trial is subjected to rigorous controls to ensurethat individuals are not unnecessarily put at risk and that they arefully informed about their role in the study. To account for thepsychological effects of receiving treatments, volunteers are randomlygiven placebo or interleukin 22 receptor inhibitor. Furthermore, toprevent the doctors from being biased in treatments, they are notinformed as to whether the medication they are administering is ainterleukin 22 receptor inhibitor or a placebo. Using this randomizationapproach, each volunteer has the same chance of being given either thenew treatment or the placebo.

[0186] Volunteers receive either the interleukin 22 receptor inhibitoror placebo for eight week period with biological parameters associatedwith the indicated disease state or condition being measured at thebeginning (baseline measurements before any treatment), end (after thefinal treatment), and at regular intervals during the study period. Suchmeasurements include the levels of nucleic acid molecules encodinginterleukin 22 receptor or interleukin 22 receptor protein levels inbody fluids, tissues or organs compared to pre-treatment levels. Othermeasurements include, but are not limited to, indices of the diseasestate or condition being treated, body weight, blood pressure, serumtiters of pharmacologic indicators of disease or toxicity as well asADME (absorption, distribution, metabolism and excretion) measurements.

[0187] Information recorded for each patient includes age (years),gender, height (cm), family history of disease state or condition(yes/no), motivation rating (some/moderate/great) and number and type ofprevious treatment regimens for the indicated disease or condition.

[0188] Volunteers taking part in this study are healthy adults (age 18to 65 years) and roughly an equal number of males and femalesparticipate in the study. Volunteers with certain characteristics areequally distributed for placebo and interleukin 22 receptor inhibitortreatment. In general, the volunteers treated with placebo have littleor no response to treatment, whereas the volunteers treated with theinterleukin 22 receptor inhibitor show positive trends in their diseasestate or condition index at the conclusion of the study.

Example 12

[0189] RNA Isolation

[0190] Poly(A)+ mRNA Isolation

[0191] Poly(A)+ mRNA was isolated according to Miura et al., (Clin.Chem., 1996, 42, 1758-1764). Other methods for poly(A)+ mRNA isolationare routine in the art. Briefly, for cells grown on 96-well plates,growth medium was removed from the cells and each well was washed with200 μL cold PBS. 60 μL lysis buffer (10 mM Tris-HCl, pH 7.6, 1 mM EDTA,0.5 M NaCl, 0.5% NP-40, 20 mM vanadyl-ribonucleoside complex) was addedto each well, the plate was gently agitated and then incubated at roomtemperature for five minutes. 55 μL of lysate was transferred to Oligod(T) coated 96-well plates (AGCT Inc., Irvine Calif.). Plates wereincubated for 60 minutes at room temperature, washed 3 times with 200 μLof wash buffer (10 mM Tris-HCl pH 7.6, 1 mM EDTA, 0.3 M NaCl). After thefinal wash, the plate was blotted on paper towels to remove excess washbuffer and then air-dried for 5 minutes. 60 μL of elution buffer (5 mMTris-HCl pH 7.6), preheated to 70° C., was added to each well, the platewas incubated on a 90° C. hot plate for 5 minutes, and the eluate wasthen transferred to a fresh 96-well plate.

[0192] Cells grown on 100 mm or other standard plates may be treatedsimilarly, using appropriate volumes of all solutions.

[0193] Total RNA Isolation

[0194] Total RNA was isolated using an RNEASY 96™ kit and bufferspurchased from Qiagen Inc. (Valencia, Calif.) following themanufacturer's recommended procedures. Briefly, for cells grown on96-well plates, growth medium was removed from the cells and each wellwas washed with 200 μL cold PBS. 150 μL Buffer RLT was added to eachwell and the plate vigorously agitated for 20 seconds. 150 μL of 70%ethanol was then added to each well and the contents mixed by pipettingthree times up and down. The samples were then transferred to the RNEASY96™ well plate attached to a QIAVAC™ manifold fitted with a wastecollection tray and attached to a vacuum source. Vacuum was applied for1 minute. 500 μL of Buffer RW1 was added to each well of the RNEASY 96™plate and incubated for 15 minutes and the vacuum was again applied for1 minute. An additional 500 μL of Buffer RW1 was added to each well ofthe RNEASY 96™ plate and the vacuum was applied for 2 minutes. 1 mL ofBuffer RPE was then added to each well of the RNEASY 96™ plate and thevacuum applied for a period of 90 seconds. The Buffer RPE wash was thenrepeated and the vacuum was applied for an additional 3 minutes. Theplate was then removed from the QIAVAC™ manifold and blotted dry onpaper towels. The plate was then re-attached to the QIAVAC™ manifoldfitted with a collection tube rack containing 1.2 mL collection tubes.RNA was then eluted by pipetting 140 μL of RNAse free water into eachwell, incubating 1 minute, and then applying the vacuum for 3 minutes.

[0195] The repetitive pipetting and elution steps may be automated usinga QIAGEN Bio-Robot 9604 (Qiagen, Inc., Valencia Calif.). Essentially,after lysing of the cells on the culture plate, the plate is transferredto the robot deck where the pipetting, DNase treatment and elution stepsare carried out.

Example 13

[0196] Real-Time Quantitative PCR Analysis of Interleukin 22 ReceptormRNA Levels

[0197] Quantitation of interleukin 22 receptor mRNA levels wasaccomplished by real-time quantitative PCR using the ABI PRISM™ 7600,7700, or 7900 Sequence Detection System (PE-Applied Biosystems, FosterCity, Calif.) according to manufacturer's instructions. This is aclosed-tube, non-gelbased, fluorescence detection system which allowshigh-throughput quantitation of polymerase chain reaction (PCR) productsin real-time. As opposed to standard PCR in which amplification productsare quantitated after the PCR is completed, products in real-timequantitative PCR are quantitated as they accumulate. This isaccomplished by including in the PCR reaction an oligonucleotide probethat anneals specifically between the forward and reverse PCR primers,and contains two fluorescent dyes. A reporter dye (e.g., FAM or JOE,obtained from either PE-Applied Biosystems, Foster City, Calif., OperonTechnologies Inc., Alameda, Calif. or Integrated DNA Technologies Inc.,Coralville, Iowa) is attached to the 5′ end of the probe and a quencherdye (e.g., TAMRA, obtained from either PE-Applied Biosystems, FosterCity, Calif., Operon Technologies Inc., Alameda, Calif. or IntegratedDNA Technologies Inc., Coralville, Iowa) is attached to the 3′ end ofthe probe. When the probe and dyes are intact, reporter dye emission isquenched by the proximity of the 3′ quencher dye. During amplification,annealing of the probe to the target sequence creates a substrate thatcan be cleaved by the 5′-exonuclease activity of Taq polymerase. Duringthe extension phase of the PCR amplification cycle, cleavage of theprobe by Taq polymerase releases the reporter dye from the remainder ofthe probe (and hence from the quencher moiety) and a sequence-specificfluorescent signal is generated. With each cycle, additional reporterdye molecules are cleaved from their respective probes, and thefluorescence intensity is monitored at regular intervals by laser opticsbuilt into the ABI PRISM™ Sequence Detection System. In each assay, aseries of parallel reactions containing serial dilutions of mRNA fromuntreated control samples generates a standard curve that is used toquantitate the percent inhibition after antisense oligonucleotidetreatment of test samples.

[0198] Prior to quantitative PCR analysis, primer-probe sets specific tothe target gene being measured are evaluated for their ability to be“multiplexed” with a GAPDH amplification reaction. In multiplexing, boththe target gene and the internal standard gene GAPDH are amplifiedconcurrently in a single sample. In this analysis, mRNA isolated fromuntreated cells is serially diluted. Each dilution is amplified in thepresence of primer-probe sets specific for GAPDH only, target gene only(“single-plexing”), or both (multiplexing). Following PCR amplification,standard curves of GAPDH and target mRNA signal as a function ofdilution are generated from both the single-plexed and multiplexedsamples. If both the slope and correlation coefficient of the GAPDH andtarget signals generated from the multiplexed samples fall within 10% oftheir corresponding values generated from the single-plexed samples, theprimer-probe set specific for that target is deemed multiplexable. Othermethods of PCR are also known in the art.

[0199] PCR reagents were obtained from Invitrogen Corporation,(Carlsbad, Calif.). RT-PCR reactions were carried out by adding 20 μLPCR cocktail (2.5×PCR buffer minus MgCl₂, 6.6 mM MgCl₂, 375 μM each ofDATP, dCTP, dCTP and dGTP, 375 nM each of forward primer and reverseprimer, 125 nM of probe, 4 Units RNAse inhibitor, 1.25 Units PLATINUM®Taq, 5 Units MuLV reverse transcriptase, and 2.5×ROX dye) to 96-wellplates containing 30 μL total RNA solution (20-200 ng). The RT reactionwas carried out by incubation for 30 minutes at 48° C. Following a 10minute incubation at 95° C. to activate the PLATINUM® Taq, 40 cycles ofa two-step PCR protocol were carried out: 95° C. for 15 seconds(denaturation) followed by 60° C. for 1.5 minutes (annealing/extension).

[0200] Gene target quantities obtained by real time RT-PCR arenormalized using either the expression level of GAPDH, a gene whoseexpression is constant, or by quantifying total RNA using RiboGreen™(Molecular Probes, Inc. Eugene, Oreg.). GAPDH expression is quantifiedby real time RT-PCR, by being run simultaneously with the target,multiplexing, or separately. Total RNA is quantified using RiboGreen RNAquantification reagent (Molecular Probes, Inc. Eugene, Oreg.). Methodsof RNA quantification by RiboGreen are taught in Jones, L. J., et al,(Analytical Biochemistry, 1998, 265, 368-374).

[0201] In this assay, 170 μL of RiboGreen™ working reagent (RiboGreenreagent diluted 1:350 in 10 mM Tris-HCl, 1 mM EDTA, pH 7.5) is pipettedinto a 96-well plate containing 30 μL purified, cellular RNA. The plateis read in a CytoFluor 4000 (PE Applied Biosystems) with excitation at485 nm and emission at 530 nm.

[0202] Probes and primers to human interleukin 22 receptor were designedto hybridize to a human interleukin 22 receptor sequence, usingpublished sequence information (a genomic sequence represented by thecomplement of residues 1710000-1734000 of GenBank accession numberNT_(—)004359.8, incorporated herein as SEQ ID NO: 4). For humaninterleukin 22 receptor the PCR primers were:

[0203] forward primer: TACCCCCACGCCAATCC (SEQ ID NO: 5)

[0204] reverse primer: GGTAGAACAGGTCATGGAAGATGTC (SEQ ID NO: 6) and

[0205] the PCR probe was: FAM-CAGGCGATGGCCACCGGCTAA-TAMRA

[0206] (SEQ ID NO: 7) where FAM is the fluorescent dye and TAMRA is thequencher dye. For human GAPDH the PCR primers were:

[0207] forward primer: GAAGGTGAAGGTCGGAGTC(SEQ ID NO:8)

[0208] reverse primer: GAAGATGGTGATGGGATTTC (SEQ ID NO:9) and the PCRprobe was: 5′ JOE-CAAGCTTCCCGTTCTCAGCC-TAMRA 3′ (SEQ ID NO: 10) whereJOE is the fluorescent reporter dye and TAMRA is the quencher dye.

Example 14

[0209] Northern Blot Analysis of Interleukin 22 Receptor mRNA Levels

[0210] Eighteen hours after antisense treatment, cell monolayers werewashed twice with cold PBS and lysed in 1 mL RNAZOL™ (TEL-TEST “B” Inc.,Friendswood, Tex.). Total RNA was prepared following manufacturer'srecommended protocols. Twenty micrograms of total RNA was fractionatedby electrophoresis through 1.2% agarose gels containing 1.1%formaldehyde using a MOPS buffer system (AMRESCO, Inc. Solon, Ohio). RNAwas transferred from the gel to HYBOND™-N+ nylon membranes (AmershamPharmacia Biotech, Piscataway, N.J.) by overnight capillary transferusing a Northern/Southern Transfer buffer system (TEL-TEST “B” Inc.,Friendswood, Tex.). RNA transfer was confirmed by UV visualization.Membranes were fixed by UV cross-linking using a STRATALINKER™ UVCrosslinker 2400 (Stratagene, Inc, La Jolla, Calif.) and then probedusing QUICKHYB™ hybridization solution (Stratagene, La Jolla, Calif.)using manufacturer's recommendations for stringent conditions.

[0211] To detect human interleukin 22 receptor, a human interleukin 22receptor specific probe was prepared by PCR using the forward primerTACCCCCACGCCAATCC (SEQ ID NO: 5) and the reverse primerGGTAGAACAGGTCATGGAAGATGTC (SEQ ID NO: 6). To normalize for variations inloading and transfer efficiency membranes were stripped and probed forhuman glyceraldehyde-3-phosphate dehydrogenase (GAPDH) RNA (Clontech,Palo Alto, Calif.).

[0212] Hybridized membranes were visualized and quantitated using aPHOSPHORIMAGER™ and IMAGEQUANT™ Software V3.3 (Molecular Dynamics,Sunnyvale, Calif.). Data was normalized to GAPDH levels in untreatedcontrols.

Example 15

[0213] Antisense Inhibition of Human Interleukin 22 Receptor Expressionby Chimeric Phosphorothioate Oligonucleotides Having 2′-MOE Wings and aDeoxy Gap

[0214] In accordance with the present invention, a series of antisensecompounds were designed to target different regions of the humaninterleukin 22 receptor RNA, using published sequences (a genomicsequence represented by the complement of residues 1710000-1734000 ofGenBank accession number NT_(—)004359.8, incorporated herein as SEQ IDNO: 4; and GenBank accession number NM_(—)021258.1, incorporated hereinas SEQ ID NO: 11). The compounds are shown in Table 1. “Target site”indicates the first (5′-most) nucleotide number on the particular targetsequence to which the compound binds. All compounds in Table 1 arechimeric oligonucleotides (“gapmers”) 20 nucleotides in length, composedof a central “gap” region consisting of ten 2′-deoxynucleotides, whichis flanked on both sides (5′ and 3′ directions) by five-nucleotide“wings”. The wings are composed of 2′-methoxyethyl (2′-MOE)nucleotides.The internucleoside (backbone) linkages are phosphorothioate (P═S)throughout the oligonucleotide. All cytidine residues are5-methylcytidines. The compounds were analyzed for their effect on humaninterleukin 22 receptor mRNA levels by quantitative real-time PCR asdescribed in other examples herein. Data are averages from threeexperiments in which HepG2 cells were treated with the oligonucleotidesof the present invention. The positive control for each datapoint isidentified in the table by sequence ID number. If present, “N.D.”indicates “no data”. TABLE 1 Inhibition of human interleukin 22 receptormRNA levels by chimeric phosphorothioate oligonucleotides having 2′-MOEwings and a deoxy gap TARGET SEQ ID TARGET % SEQ CONTROL ISIS # REGIONNO SITE SEQUENCE INHIB ID NO SEQ ID NO 249270 5′UTR 4 164gggctggcacagagccctcc 50 13 1 249271 Coding 4 208 agccagggatcccacagtca 6014 1 249272 Coding 4 4575 cacgtgctggagcagatccg 66 15 1 249273 Coding 44595 aagttgctggactggaattt 71 16 1 249274 Coding 11 190ctctccgtacgtcttatact 71 17 1 249275 Coding 4 5984 atccgctgacagcccttctt75 18 1 249276 Coding 4 6011 accgtcaggttgcaggactt 71 19 1 249277 Coding4 6041 tagtagagctccgtgaggtt 70 20 1 249278 Coding 11 369ttgagggtagtgtgctgcag 58 21 1 249279 Coding 4 8996 tagaacaggtcatggaagat59 22 1 249280 Coding 11 551 gcttccctccaaggtgcatt 78 23 1 249281 Coding4 15003 ggccgaagaactcatattct 88 24 1 249282 Coding 4 15035gatggtgccaaggaactctg 79 25 1 249283 Coding 11 677 ggtctggcagtgtcttcact45 26 1 249284 Coding 11 684 catgtccggtctggcagtgt 56 27 1 249285 Intron:4 19838 gagtaggtccatgtccggtc 0 28 1 exon Junction 249286 Coding 4 19858acaggaaggctccggagaag 59 29 1 249287 Coding 4 19863 ggagaacaggaaggctccgg62 30 1 249288 Coding 4 19871 aagcccatggagaacaggaa 73 31 1 249289 Coding4 19897 tcaggtagcagagtactgcg 77 32 1 249290 Coding 4 19909catatctgtagctcaggtag 73 33 1 249291 Coding 4 21584 cgctgaggtcaaagacaggg77 34 1 249292 Coding 4 21634 agacaccctgatctgggagt 71 35 1 249293 Coding4 21658 agctcctgcgggctccctgg 64 36 1 249294 Coding 4 21663tgtggagctcctgcgggctc 54 37 1 249295 Coding 4 21676 caggctatgccgctgtggag89 38 1 249296 Coding 4 21693 aagtaggtgatctcggacag 4 39 1 249297 Coding4 21706 gtctggctgccctaagtagg 75 40 1 249298 Coding 4 21820ggtcacctgaggtgcatagg 74 41 1 249299 Coding 4 21908 agctgtccggagtggcttga71 42 1 249300 Coding 4 21926 ccccataggagggaggccag 45 43 1 249301 Coding4 21932 tgcataccccataggaggga 66 44 1 249302 Coding 4 21941aaccttccatgcatacccca 60 45 1 249303 Coding 4 21953 agtctttgccagaaccttcc75 46 1 249304 Coding 4 21981 ttaggactagaaagtgtccc 38 47 1 249305 Coding4 22031 agcttccagctggtggctct 17 48 1 249306 Coding 4 22041cctaacatgcagcttccagc 85 49 1 249307 Coding 4 22059 tcctgcagagaaaggccacc89 50 1 249308 Coding 4 22079 ccatagccaaggaggtcacc 77 51 1 249309 Coding4 22134 ctgtctgtgcaaatccccag 94 52 1 249310 Coding 4 22158tgtagcacatttgggtcaga 74 53 1 249311 Coding 4 22198 ctggccctttaggtactgtg79 54 1 249312 Coding 4 22234 gtggccctcgatctggactg 82 55 1 249313 Coding4 22319 gggactccagcaggccccag 86 56 1 249314 Coding 4 22345cttggcttcatccttgggac 59 57 1 249315 Coding 4 22379 gctgctccaggtctgaggtc75 58 1 249316 Coding 4 22414 ggccaggcctctgaaaagag 84 59 1 249317 Coding4 22431 tcccactgcacagtcagggc 88 60 1 249318 Stop 4 22450ttcccattcccctcaggact 64 61 1 Codon 249319 3′UTR 4 22575ctccctctgcttctctcaag 86 62 1 249320 3′UTR 4 22616 tccggtgaggagcgcaccca53 63 1 249321 3′UTR 4 22680 gcgcttgtctacacaagctg 65 64 1 249322 3′UTR 422741 ggagtttccctgcatttcct 86 65 1 249323 3′UTR 4 22793ttccctgagcactttgaatc 78 66 1 249324 3′UTR 4 22841 tcgagctagattgtgaaact73 67 1 249325 3′UTR 4 22920 cttttccaggcctggttctt 43 68 1 249326 3′UTR 422949 ttctggttctgcccagcctc 74 69 1 249327 3′UTR 4 22964cagaagtgcaggttgttctg 73 70 1 249328 3′UTR 4 23029 ctgggaatgagctgcaggcc 071 1 249329 3′UTR 4 23045 caggcagttgccctggctgg 88 72 1 249330 3′UTR 423084 ttgttctatcagaggaatga 67 73 1 249331 3′UTR 4 23112tccctccctggtggacctgc 56 74 1 249332 3′UTR 4 23162 ttctcaggatagggtctgaa26 75 1 249333 3′UTR 4 23262 gggctgaacccaaggcagag 80 76 1 249334 3′UTR 423398 atcttcaccacaactccatg 57 77 1 249335 3′UTR 4 23411atgacttcatttcatcttca 79 78 1 249336 3′UTR 4 23446 cccatgtaccaggcactatt83 79 1 249337 3′UTR 4 23464 accgtttattgggcactgcc 77 80 1 249338 3′UTR 423472 aaatagctaccgtttattgg 53 81 1 249339 Intron 4 4278cattccataaatgtcaccac 55 82 1 249340 Intron: 4 4677 agaagcctacgtcttatact63 83 1 exon junction 249341 Intron: 4 5949 ctctccgtacctgcaggtca 75 84 1exon junction 249342 Intron 4 7634 tttaactgactcacagttcc 68 85 1 249343Intron 4 8253 cctcatttaccctgaatcta 27 86 1 249344 Intron: 4 15112ccgaactcacctggcagtgt 70 87 1 exon junction 249345 Intron: 4 19951gatggctcaccagggagttg 43 88 1 exon junction 249346 Intron 4 20141ccaccccacttcaatgtgtc 55 89 1 249347 5′UTR 4 148 ctcccttggcctctactctg 8890 1

[0215] As shown in Table 1, SEQ ID NOS 14, 15, 16, 17, 18, 19, 20, 23,24, 25, 30, 31, 32, 33, 34, 35, 36, 38, 40, 41, 42, 44, 45, 46, 49, 50,51, 52, 53, 54, 55, 56, 58, 59, 60, 61, 62, 64, 65, 66, 67, 69, 70, 72,73, 76, 78, 79, 80, 83, 84, 85, 87 and 90 demonstrated at least 60%inhibition of human interleukin 22 receptor expression in this assay andare therefore preferred. More preferred are SEQ ID NOs: 38, 52 and 72.The target regions to which these preferred sequences are complementaryare herein referred to as “preferred target segments” and are thereforepreferred for targeting by compounds of the present invention. Thesepreferred target segments are shown in Table 2. The sequeces representthe reverse complement of the preferred antisense compounds shown inTable 1. “Target site” indicates the first (5′-most) nucleotide numberon the particular target nucleic acid to which the oligonucleotidebinds. Also shown in Table 2 is the species in which each of thepreferred target segments was found. TABLE 2 Sequence and position ofpreferred target segments identified in interleukin 22 receptor. TARGETSEQ ID TARGET REV COMP SEQ ID SITEID NO SITE SEQUENCE OF SEQ ID ACTIVEIN NO 165767 4 208 tgactgtgggatccctggct 14 H. sapiens 91 165768 4 4575cggatctgctccagcacgtg 15 H. sapiens 92 165769 4 4595 aaattccagtccagcaactt16 H. sapiens 93 165770 11 190 agtataagacgtacggagag 17 H. sapiens 94165771 4 5984 aagaagggctgtcagcggat 18 H. sapiens 95 165772 4 6011aagtcctgcaacctgacggt 19 H. sapiens 96 165773 4 6041 aacctcacggagctctacta20 H. sapiens 97 165776 11 551 aatgcaccttggagggaagc 23 H. sapiens 98165777 4 15003 agaatatgagttcttcggcc 24 H. sapiens 99 165778 4 15035cagagttccttggcaccatc 25 H. sapiens 100 165783 4 19863ccggagccttcctgttctcc 30 H. sapiens 101 165784 4 19871ttcctgttctccatgggctt 31 H. sapiens 102 165785 4 19897cgcagtactctgctacctga 32 H. sapiens 103 165786 4 19909ctacctgagctacagatatg 33 H. sapiens 104 165787 4 21584ccctgtctttgacctcagcg 34 H. sapiens 105 165788 4 21634actcccagatcagggtgtct 35 H. sapiens 106 165789 4 21658ccagggagcccgcaggagct 36 H. sapiens 107 165791 4 21676ctccacagcggcatagcctg 38 H. sapiens 108 165793 4 21706cctacttagggcagccagac 40 H. sapiens 109 165794 4 21820cctatgcacctcaggtgacc 41 H. sapiens 110 165795 4 21908tcaagccactccggacagct 42 H. sapiens 111 165797 4 21932tccctcctatggggtatgca 44 H. sapiens 112 165798 4 21941tggggtatgcatggaaggtt 45 H. sapiens 113 165799 4 21953ggaaggttctggcaaagact 46 H. sapiens 114 165802 4 22041gctggaagctgcatgttagg 49 H. sapiens 115 165803 4 22059ggtggcctttctctgcagga 50 H. sapiens 116 165804 4 22079ggtgacctccttggctatgg 51 H. sapiens 117 165805 4 22134ctggggatttgcacagacag 52 H. sapiens 118 165806 4 22158tctgacccaaatgtgctaca 53 H. sapiens 119 165807 4 22198cacagtacctaaagggccag 54 H. sapiens 120 165808 4 22234cagtccagatcgagggccac 55 H. sapiens 121 165809 4 22319ctggggcctgctggagtccc 56 H. sapiens 122 165811 4 22379gacctcagacctggagcagc 58 H. sapiens 123 165812 4 22414ctcttttcagaggcctggcc 59 H. sapiens 124 165813 4 22431gccctgactgtgcagtggga 60 H. sapiens 125 165814 4 22450agtcctgaggggaatgggaa 61 H. sapiens 126 165815 4 22575cttgagagaagcagagggag 62 H. sapiens 127 165817 4 22680cagcttgtgtagacaagcgc 64 H. sapiens 128 165818 4 22741aggaaatgcagggaaactcc 65 H. sapiens 129 165819 4 22793gattcaaagtgctcagggaa 66 H. sapiens 130 165820 4 22841agtttcacaatctagctcga 67 H. sapiens 131 165822 4 22949gaggctgggcagaaccagaa 69 H. sapiens 132 165823 4 22964cagaacaacctgcacttctg 70 H. sapiens 133 165825 4 23045ccagccagggcaactgcctg 72 H. sapiens 134 165826 4 23084tcattcctctgatagaacaa 73 H. sapiens 135 165829 4 23262ctctgccttgggttcagccc 76 H. sapiens 136 165831 4 23411tgaagatgaaatgaagtcat 78 H. sapiens 137 165832 4 23446aatagtgcctggtacatggg 79 H. sapiens 138 165833 4 23464ggcagtgcccaataaacggt 80 H. sapiens 139 165836 4 4677agtataagacgtaggcttct 83 H. sapiens 140 165837 4 5949tgacctgcaggtacggagag 84 H. sapiens 141 165838 4 7634ggaactgtgagtcagttaaa 85 H. sapiens 142 165840 4 15112acactgccaggtgagttcgg 87 H. sapiens 143 165843 4 148 cagagtagaggccaagggag90 H. sapiens 144

[0216] As these “preferred target segments” have been found byexperimentation to be open to, and accessible for, hybridization withthe antisense compounds of the present invention, one of skill in theart will recognize or be able to ascertain, using no more than routineexperimentation, further embodiments of the invention that encompassother compounds that specifically hybridize to these preferred targetsegments and consequently inhibit the expression of interleukin 22receptor.

[0217] According to the present invention, antisense compounds includeantisense oligomeric compounds, antisense oligonucleotides, ribozymes,external guide sequence (EGS) oligonucleotides, alternate splicers,primers, probes, and other short oligomeric compounds which hybridize toat least a portion of the target nucleic acid.

Example 16

[0218] Western Blot Analysis of Interleukin 22 Receptor Protein Levels

[0219] Western blot analysis (immunoblot analysis) is carried out usingstandard methods. Cells are harvested 16-20 h after oligonucleotidetreatment, washed once with PBS, suspended in Laemmli buffer (100ul/well), boiled for 5 minutes and loaded on a 16% SDS-PAGE gel. Gelsare run for 1.5 hours at 150 V, and transferred to membrane for westernblotting. Appropriate primary antibody directed to interleukin 22receptor is used, with a radiolabeled or fluorescently labeled secondaryantibody directed against the primary antibody species. Bands arevisualized using a PHOSPHORIMAGER™ (Molecular Dynamics, SunnyvaleCalif.).

0 SEQUENCE LISTING <160> NUMBER OF SEQ ID NOS: 144 <210> SEQ ID NO 1<211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400>SEQUENCE: 1 tccgtcatcg ctcctcaggg 20 <210> SEQ ID NO 2 <211> LENGTH: 20<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223>OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 2gtgcgcgcga gcccgaaatc 20 <210> SEQ ID NO 3 <211> LENGTH: 20 <212> TYPE:DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 3 atgcattctgcccccaagga 20 <210> SEQ ID NO 4 <211> LENGTH: 24001 <212> TYPE: DNA<213> ORGANISM: Homo sapiens <400> SEQUENCE: 4 tgaattccag agcacacagggccaggacac ctttgtaccc ctgctcccca ccggcacagg 60 tcccacccac ccccttctccccgccccatg cccgggtgga gccagctgcc agggcgccaa 120 gactcccagc cctctctctgtgcacggcag agtagaggcc aagggagggc tctgtgccag 180 ccccgatgag gacgctgctgaccatcttga ctgtgggatc cctggctggt gagtgcccat 240 ctaccctgtg gtcagacctcccttatccca ggggccaacg atcagggtca tctccccagc 300 accctcccca gcccgagcatagccagtttc agtttctccc ctgctagggc tgagcagggt 360 gccgggtgtt cacggagaagcaaatgccca gcttagtttg ctgggggcat gagagggtgc 420 ctcagaccct gggttcttccagggacccaa aggaccctcg ggtcttgagg gatatagcca 480 gctgtctgaa ctgggggtgctggatcgctg cacctcagtg aatggagaat tggcctggag 540 gaggcagaca tccttcggtccctcaaacgg tgtcggcaca gtttgtacca tccttcctct 600 gcctctcact tggcactggcctgacagggg aggaggagga aggaccgggg gaaacgaagc 660 ttgagttcac tattgcctccctccatgccc ctgcagggcc tctttggccc cttttgttat 720 catagatctg aaagagcattttctcctaga gtggccaggt ctgctacatt aataggtctt 780 aagacacagg actcatttggaaacaggtct taagggcata gtgttcaaag ccaataaagt 840 gagcggagtg gggggctcatcattatggag aacgttacat gagaccctag actctctctc 900 tgctctcctg gtgcattgcatgaaattcag ggaacttcct taggaagtca aagcatagtc 960 ctgattgagg atggcgagcaggtatggcct cgaccaggcc ctccttgctc aagtgcagga 1020 cgaggcccac gaaaaggcagagcttcccac cttctctgcc actgtcccgc cagggctgcc 1080 tgcgtcccca gggcttccagctctgtggga ccccctccag tctgcttccc agcatcaagc 1140 tgggcgttcc aggaagatgtgaggcacccc cttgaggcac caagacacgt ccctttagac 1200 cctcagccat actggttagaaatcctagag gctcccagcc ctttgagatg ggggacccca 1260 agaaatagcc cttaggagattgtcaagggt cagctgcctc gcgctgcaca caccgggccc 1320 tcacccgccc aatcttaggctgactccaga gcatgtggaa ttgtttcttg tttgtgtatt 1380 tttccttagc ttcccagctggttgacagaa cattgttttt ccacatgaat gtgaaaaaac 1440 aagttgcctt ttataaatccgccaggctgc caggctccac acaacacatg tctccccaga 1500 tttcacaata tagactttcacccgggaagt gccgaagcaa cacaggctga ggaatggatg 1560 atagatttga ggaggaaaaggagcgaggag ggtgggagaa gagggggaga tcttgttaga 1620 ccctgacatc cctaaaagacaggagtgggc ctaggttcac ttaccccact tcaggaagct 1680 aactgcagtt ccctcctgaaaagtggtgcc aacctccacg tagcccaggg tcctttgcct 1740 tggcaacatg cccccacctcagccaccagc ctccttgcaa cccagatggg ccctctattt 1800 cccacccaga acactgacctcaaccagcaa ccgttcccgc tgcagactct accccaagcc 1860 ttgatggagc ctggcttctcaaagcccacc ctcacccttc tgccctgacc caccacccaa 1920 ccagaagcca ctgttcaggtctgaccttga ctggactcct ttcctcaact ttagactcag 1980 ccctgaatcc aggacccctccctccagaac tgaccccaat tccacccagc ctcacatcca 2040 aacaagatgt cagctacatcctaaggctcc acggcacagc cgtcaccttg cgtagaggca 2100 gtagacccct ccccgcgccatccctaaggc cccattctgg ggactccagg aagcctgaag 2160 acattttgca gctgctgggccagaacccaa attcagagag gagggcagcc ttcgaggttg 2220 gccagaggat gtgcagcggggtcggaccat tcttccgtcc ccatcggtcc tcgccaagcc 2280 tgaggacccc tcttgtgtgtagagttggag taggggaatc aataactgtc tgctccctca 2340 caccttgggc ccactggcagtcgtagtcgt gtctggggaa ggagaggcac agacatattc 2400 aagacacaca cccaggccgcaaggccttga cccagctggg gttgcttaac attttcccac 2460 ctgtttattt cccacgccagccggccaggg cctccgagct ccgagccagc tgccagcttg 2520 ggccgccatt ttcccgcaggctcttgctct ccacagccca agcctggggg cctgagggaa 2580 gggttttata agttaaggactctttgttta cagtgcagat tgctttagag gctgcagccc 2640 ctccccagcc ccttccttgaggctcaggcc ctcactgccc ttcccttaac agaagcagaa 2700 gtgggctgga caaagacctagcctggaaga gagggctgtg gactcttcaa tggccagctt 2760 ctcttctcct ctctcttctcctcctcccta aattcatccc tttatcaggt gctcaggtcc 2820 ctgaagcccc ccaccctcatataaaagggt gaacaagata gccccctccc tacagagcga 2880 atgtcttccc ttcccaggtctcctcccttc agactcaaca catgtacata ggggcctgtt 2940 agacactaaa cacattcacagacgccaacc cgctctatcc gccccacaac cagtctagca 3000 ctgggatgaa tattgtaggttcttcacatg ctaactgtga tgctgggcgg cagctctctc 3060 tgagcctcag ctcattcatcaaatggggat agtgatagtg ggcacgttgt cgtgaggatt 3120 taatgaaggg agggacttgcctgaaagagg ctgtgcactt cacaaatgcc agtggctgct 3180 gttgtcattg ttgtttgtctttcggtaaat caaactatat ccattttaga gatgaggcaa 3240 ttgaagttct cgttgcttaaacaactttcc caaggccacc ctgcccacgt gtgggctcag 3300 tcaagagcgc ctcctccaccattgcttcat ctgatcaact tggggcaagt cacttcttac 3360 acactttagt tacatatctctaaaataagg gtgaggacac caacttcagg ttgtgttaaa 3420 aagtgaatga gagacaataccaagcatcaa tggcgatgtg gagaaactga acccctcaaa 3480 caatgctggt gggaatttaagatggtgcag ccataacatc ctggacaata tatgagaccc 3540 catcactaca aaaaatttaaaaataaatta gacggatgtg gtggtgcatg tctgtagtcc 3600 cagctacaca ggaggctgaggtgcgaggat tgcttaagcc caggaagtcg aggctgcagt 3660 gagccatgat cacgccactgcactccagcc tgggggacag agcaagaccc tgtctcaaag 3720 accaaaaaaa tggtgtggccactttagaaa atagttcgac agttcttcaa aaggttaaag 3780 atagagctac tatataacctaccaaattcc actcttagat atatacccaa gacaactgaa 3840 aacatatgtc cacttataaaattgaacacg gtgttcacag cagcattatt catggtagcc 3900 aaaaagtaga aacaacccaaatgtcctgat gagtggataa acaaaatgtg gtatatcaca 3960 caatggaata ttattcagccctaaaaagga agtactaatc catgctacca cacgtgtgaa 4020 ccttgaaaac attatgctaagtgaaagaag ccagccaagc tctatttaca taaaaagtct 4080 agagtaggga cattcacagacacaggaagt agattcgtgg ttgtcagggt ttaggggagg 4140 agaagaatgg gtggagtgactgctaatggg catgcagttt ctttttgggg tgctaaaaat 4200 gttctggaat tagataatagcgatgattgc acaacgttgt gaaaatgcta agaaaccatt 4260 gaactgaaca ctgtaaggtggtgacattta tggaatgtga atcacaaccc attgcaaagc 4320 aaaatggctg agaatggatggttggttaga acagcgtggg gagcatcccc tcttgctggt 4380 ccatttgtag aaggctgggggccagggtct ccctgcccac ccaccaggct ggcatcttga 4440 aagcaaaggc actttgcatgaaatttatat ggggcaggac tctgactgag cccatacatg 4500 ggcagggtga ccctgggaaagcccctgctc accctcctcc caccctcctg cagctcacgc 4560 ccctgaggac ccctcggatctgctccagca cgtgaaattc cagtccagca actttgaaaa 4620 catcctgacg tgggacagcgggccggaggg caccccagac acggtctaca gcatcgagta 4680 taagacgtag gcttctctcttgaccccacc tcctttgact gatctgaagc cagcccctct 4740 ccaaggaatt cccctcctctccatatgttc ccatgtcccc accaggactg gtgtcacata 4800 gcccctccca gataaagctttgctccttta tgtttctttt gcagagggag aataatataa 4860 gggtaaaaag tacaggtgatagggtcaggc tgtctgggct taaatgccgc tctcgcctgt 4920 ataaccttgg gcaggtaacctcacgaagcc tcagtttctc cacccacaaa gtgaggacaa 4980 tgatagcact ttcatcacagggttgttggg tgggttaaac aagataaagt cattagcact 5040 tgtgcctgga aaattataagctaccaataa ttgttttatt attattagtg agactaggtc 5100 aaccgaagat gcacccagcccctctagccc acgctcctgt tcctgctcct ctccctccca 5160 tgcaggtgag gaggggtggcctgggctcag gagtcctcaa acctggcttc cactcctggt 5220 catgccacgt tgtcctgtagcaagtcactt ccccctctct gagcctcatt tctctcatct 5280 gtaaaatggg gataacagggcccacatcaa aggactatgg tgatgacgag cccagaatgt 5340 ggactcagac tggctgggttcaaatcctgg ctctccacat gcacacttca aacacgtggg 5400 caatttactt ctttgagcctcatcttcttt gtttgaaaaa taaacagaat aggctgggca 5460 cagtggctca cacctataatctcagcactt tcagaggctg aggcaggtgg atcacttgag 5520 gttaggagtt cgagaccagcctgaccaaca gggtggccat actgtctcta ctaaaataca 5580 aaaattagcc aggcatggtggtgcactcct gtaatcccag ctactcggga ggctgaggca 5640 ggagaatcac tcgaaccccagaggtagaag ttgcagtgag ccaagactgc accactgcac 5700 tccagcctga gtgacagagcgaggctgtgt ctcaagaaaa aaaaaaaaaa agaaaggaag 5760 gaataaagaa agaaaaagagaagaatgcat atgcctcaga gaatcattgt gagaacaaaa 5820 tgagatcatt cgcataaagggcctgggcca ctgaacgtgc taagtaaata tcagatctcc 5880 ttggcattat taataagccacaggcctagc gctggccttt tcatcaccac ggtgacttgg 5940 ccccttcctg acctgcaggtacggagagag ggactgggtg gcaaagaagg gctgtcagcg 6000 gatcacccgg aagtcctgcaacctgacggt ggagacgggc aacctcacgg agctctacta 6060 tgccagggtc accgctgtcagtgcgggagg ccggtcagcc accaagatga ctgacaggtt 6120 cagctctctg cagcacagtgagttggagcc cctgtgggtt tcaggggagg gaagaggaaa 6180 gcgcaggtgt ttgcggggtgggatggcctc ttcccctctg gagtgggtgg agggtccaga 6240 aggggctctc tgcaaggggaggggcagctg gggatgtggc ttttagggaa gccccccacc 6300 cccaccaatt acagtgtgttcctagcaggt ttgttcactt ttctccacct ccaccccaac 6360 ctccctagtt cagaccacccttatcttgac ccccgactgc agtcaaggcc acctaacttg 6420 tttccccaac tctcctctttaccacctcct ccagcccatt ctccacagag catttagaga 6480 gatctcctca acacataaatcccaccttct cttctcagct taaaaccctc gggcagcttc 6540 tcatgactct acaaataagatccaactcct tgccacggcc cacgaggccc tgcctatcct 6600 ggtcctcatc ttgaacatgtcttcccaatg catcgagcag ccaagcttct gacagtccct 6660 gcctcgggtt ccttgctgctcccactctct gggggcactt ctgagctctt cacatgactg 6720 gttcttttat agtcctcacggctcagcttc aacatcctaa gtcagtgtcc ccacctccag 6780 ccctgatgac ctgatttctgtaggccacca tctgtgattc tctagtgcaa tacagggctt 6840 attgtcttca tcgctcagatcacacgctgc aattactgtg tttgctcaca catgttatca 6900 tctgcctccc cctctaaaatgtgagctcta gaactgtatg tgtgggcggc agaatccact 6960 gcattcccag ccctcagcatagtgcctggc acatagaaga ttctcaatag ccaggcgtgg 7020 tggctcacac ttgtaatcccagcactttgg gaggctgagg caagaggatc acttgagcct 7080 aggagttcaa gaccagcctggggaacacag tgagacccca tctctacaaa aaatttaaaa 7140 attagctggg tgtggtggcacacacttgta gtcccagcta ctcaggaggt tgaagtggga 7200 gggtagcttg aacccaggaggtcaagactg cagtgagctg ggattgcccc actgcactcc 7260 aacttaggtg acacagtgagatcctatctc aaaaaaataa aaaagatatt cagtaaatga 7320 atggttgagt ggacagcagttgctgatatg gtttggccat gtccccaccc aaatctcatc 7380 ttgaattgta gttcccataatcctgtcttg tgggaaggac ccagtaggag gtaactgaat 7440 gatggggacg gttccccccatgctattctc atgatagtgg gtacgttctt gtgagatctg 7500 atggttttat aaggggcttccccctttgct tggttctcat tttccttcct gccaccatgt 7560 gaggaagaac atgtttgcctcccattttgt aagtttcctg attgtaagtt tcctgaggcc 7620 tccccagcca tgtggaactgtgagtcagtt aaacctcttt cctttataaa ttacccagtc 7680 tgaggtgtgt ctttattagcagtgtgagaa tggactaata tagttgccaa actccccaca 7740 caagctaagc gtgtgacgatgttcaccaac tgactttata gtcacacata gctttttaag 7800 aaaataaaaa ataaaattaagtaaaatatt gtttttttaa cctggcttgt tcagtgaatt 7860 cacattcatt gtagaaaatgtgtgaaatat agaaatatac aaagaaaaat aaaagtcacc 7920 caaaatgatc ttaacattgtaacataactg cagatactag cagccgtatc tactttgcaa 7980 tctggcattg cttttttgtcttaggttctt tttctccata tattggtatc atgaacattt 8040 acccatatca ttaaacattttgcacaacaa gatatttcac agtgcaaaat aatccattct 8100 agggagatac tctaatttacttatgtattt ccctttttga ggacatacag ctcagttttt 8160 caattttatt agcagcactgaagtaaacat tgttctgact cagatatctt caatcttgga 8220 ggccaaagga tcattgaggaatttttacaa attagattca gggtaaatga ggtccatgaa 8280 tccctgaagt tggatgcaagatttttgtat atatacattt tggcaggaag aagacccaca 8340 gcattcatca cattctcaaaggagtctgta aaccagcaaa ggtaatttgg ttatttggaa 8400 aattccatct tgagagagacagaagtcttc agcctgatct ttgaggctgc atcaatagta 8460 ttaggcccca aacattatttaggatgtgta gttgatgtca ttgacatccc caaagctgtc 8520 atactcaata gatgagggaggaagggtgct ctaggctgac ttaaggtggg gacgcagggg 8580 gctgggatgt gtgggggcaagccaaaggca tgaccaagct aggggactgg tgcccctccc 8640 cctgagttct tctcatccactcttggtcat ccctttgcca gcctgaaacc caaacaccag 8700 ggcttgtaga aacttttctcttttcactgt ttgtttagcc gttgctttca gaagcatagc 8760 tggcactgga gtggaaggagagagggagca gtcattttcc actgtccaag gttgcctcag 8820 acactaccca ccgattctgactttttcttt tctcttttct ctctccctat ccccagctac 8880 cctcaagcca cctgatgtgacctgtatctc caaagtgaga tcgattcaga tgattgttca 8940 tcctaccccc acgccaatccgtgcaggcga tggccaccgg ctaaccctgg aagacatctt 9000 ccatgacctg ttctaccacttagagctcca ggtcaaccgc acctaccaaa tggtgagtgt 9060 atgttgcacc ctggtctttctctgcctagg aagcctcttc cctcccaatt agatctgagt 9120 tgctttaaga aaaaaaggggacatgttatg taaattagca tttcccaaaa catgtccctt 9180 gagaggctcc ttgaagcctggaaatcatca caaatgacaa cttttctctt gtagattcac 9240 acacacaata gcatgctaaaagctctgaca agtcctgcag agccaagact ccactatcaa 9300 tttgttttgt tgttttgttttgttttttat ttatttattt ttattttatt attattatac 9360 tttaagtttt agggtaaatgtgcaggttag ctacatatgt atacatgtgc catgctggtg 9420 tgctgcaccc attaactcgtcatttagcat taggtatatt atcaatttgt ttaatacaat 9480 ttctccagaa tttatttgaccagagaagct gctttttttg cataatattt acattgtttt 9540 ttgtttaaca ccttctcacatcctggggaa caacctctat taatatcttc agatgttcac 9600 attagaaaat gctagaatcaatttctactg ctgcagtagt tttccaatat ttgcaggaac 9660 tatatacagg caatgtggaaggatgagtaa taataagagg tggaaggcaa gggcatacat 9720 tctcaaatag tacatgaattatttcttttc gtattcagaa aatatttggt accccctacc 9780 ctcagcatgt gccaccaatccacaattcag ttaaatgaat atttatttcc tactttgtgg 9840 tttctgcatg caaggagcttataatctagg gggtgaaaag gaagttaaaa caagaattca 9900 aatagctata ggtagaggaaaatatgggta ttgggaggtg gggcaggatg tcagcacagc 9960 aagatttgaa agacttccagaaaactttta ggaagccaaa aatgacataa tatgcatata 10020 ttacctggca tgccgcagaaattggtactg gcttccagaa agcctaatat gcataatatg 10080 catattatgt cattaatagtcatatattac ctggcatgcc acagaaattg gtattggctt 10140 tctggaagcc agtatcaatttctgaggcat ccaggtaata catgcatatt atgtcattta 10200 atcttcacca cggccttgtgaggcaggttc tatcatctcc attgtctaga tgagggaacg 10260 aagagaagtt aaaagtcacttgctcagatt gttcagcaaa taatagggta gacaggattc 10320 aaatccagct ctgtttagcccacatttctc ccaccactgg cctgcctata ctgccccacc 10380 atgatcagag agaaaagcagcaaaacttaa gccatttaaa aaaaagagag acggggcctc 10440 actgtgttgc ccaggctggtctcaaactcc tgggctcaag agatcctcct gcctcagcct 10500 cccaaagtgc tgggttgacaggcatgagcc accgcacccg gccaacttaa gacatttttg 10560 aaaagcacag aactgcccactttgacaaga gcataaagac atgtaaaaca gtttcttaaa 10620 acatggtccc agccaggcacggtggctcac gcctgtaatc ccagcacttt gggaggccga 10680 ggcgggcgga tcacgaggtcaggagatgca gaccatcctg gctaacacgg tgaaaccctg 10740 tccctattaa aaaatacaaaaaattagccg ggcatggtag tgggcgcctg tagtctcagc 10800 tactcaggag gctgaggtagaagaatggcg tgaacccggg aggtggagct tgcagtgggc 10860 caagatcgcg ccactgcactccagcctggg cgacagagca agactccatc tcaaaaaaaa 10920 aaaaaaaaca cacacacaaaaaccatggtc ccaaacaaac agcatcagaa tctcctgcag 10980 cacttgttta aaatgcagattcttgggtcc ccttccagtc aacttcatca agttattgca 11040 cttgcatccc tgggatctgaattttagtga gcaacccagg cgcttcttcc ccaacgcaaa 11100 gcttgagaac ctctgacgtggagggacccc gggagataaa cctcagaaga ctggatggga 11160 ctatgctgta gaagggatagggtggtggag gggacgaatt tttgtttctt tcggtagttg 11220 ggggagccac tagagattactaagcaggga gagtccttat cacaactatt ttttggtaaa 11280 tatttagggt cttattaaatattttattgt tgatttctaa tttaattcaa tttgtggtca 11340 gagaacacac gctgtatgatttcagtcctt ttaaatttat cgaggcttgt gttatagccc 11400 agcctatggc ctgtcctggtgaacgttcct tgtgcacctg aaaagaatgt atattttgca 11460 gttggttggt ggagtgcgctataaatatca attaggtaaa ggtgattaat tgtgttattc 11520 agatcttcta tgtctttcctgatttaggtg agaggtcaat tggtcgttta attgttgaaa 11580 atagtaaact ctccaactataattgtggat ttgtctattt tccctttaat tctgtgcatt 11640 tttgcttcgt gcattttgaaactcagttat taaatacata tatatttatg attgttgtat 11700 cttcctgata gattgaaccttctgtcatag gaattgttcc tttttatttg tagtcatact 11760 ctttgtattg aagtctactttatcgatatt aatatagaca ctccagcctt cttatggttt 11820 gcataatata tgtttttacattcattttca atctgcctct gttattatat ttaaaatgta 11880 tctcttgcag acagctctgacagcctttac ctttttttga cattaattca catgccataa 11940 aattcattct tttaaagcatacaattcagg tgttacttgc ctattcacaa agttgtgaaa 12000 ccatcaccac tatctaattctcccttatca atctttcttc aagttctcta ttttttctgt 12060 catttccaaa atgtttttgagataattcag tgggttttta aatttcagat aataatatct 12120 agaatttttc aactctaaagttcctagatt ttatagtttc catattctac tgagattttc 12180 tattcattca ttcaatatgagcttattttg tttcacttcc tggagcattg ttataccgct 12240 gctttaaaat cattgttggccaggtgaggt ggcccacgcc tgtaatccca gcactttggg 12300 aggccaaagt gggtggatcacttgaggtca ggagttcaag accatcctgg ccagtgtggt 12360 aaaaccccat ctctactaaaaatacaaaaa tcagctgggc atggtggcag gtgcctgtaa 12420 tcccagctac tcaggaggctgaggcaggag aatcatttga acctgggagg cggaggttgc 12480 agtgagctga gattgcaccactgcactcca acctgggtga cagagtaaga ctgtgtctca 12540 aataaaataa aatcattgtctgctaattca acattttggt catcagggtc agtcactatt 12600 atcttttctc ttgaatatgggtcgcatatt ctttctttgt atgtctaaca attttgaaat 12660 gtatcctgga cattgtgttgaaattactgt agattctgtt atattcctcc aaagagtgtt 12720 ggtttggttt catccggttttggtaggcaa ttaatttggc tgaactccaa actctgaaat 12780 ctctcttctt tttttgttggttaactttac ctgggctgct tggagtctac cccattcatg 12840 cctactttag gggccggtcagggatttgag cagagtttat atacagagtt tgggcctcct 12900 tctctacact ctcccctgtctgtgctttct cccctcattt tccagctgct gtgattattc 12960 caagccctgc cttttgggtattcaagccac tgagaataaa gggtttctgt ctaggtttag 13020 ccattccaca gggtgtagactgaggtctgc cttcaggcta caagacagta aaaatgggaa 13080 atgtacccat cgacatttctcctctctcca atatatgcct gcttttattt tctctccaat 13140 gcttttaagt agttggttttcattttgctt agaatttata gttgttattt tggggatgtt 13200 tggtccaata gaagatataaattactagga ttagaatgaa gaaagatcaa atatggaaat 13260 ggaagagata tcaggctggcaagtcaaaag attattgcaa tggtataggc tggaaatgag 13320 ggcctgaacc aggatggaggtcgtgaaaaa cttgggaaag gaggaatcag ggccttggca 13380 actctcctgg agttatgtctcatgatatgt aatgtgtcca gttccaacca catgagttcc 13440 acaaaacaat aagttaaaaaatttatttat gtacttcttc actcactgtc accaccatcc 13500 ccataaacac acacaaaagaaaacagtaaa ttaccctcaa ccttggcctc catgtgcctg 13560 aaaaagagaa ggcagagctgaaatacaaag agccaaagaa aattctcagt ttttgaactc 13620 ccgagaacac aggttctggccctttaaagg aagatttctg gatcctttaa gggcaatttg 13680 ataccaaaga tatgagaggggatttgtcca aaatggcttc aaggtttagg ccctggagat 13740 tagaagaaga gtgatgctattatcaaaatt agagaatcaa aagcagattt gggtttgggg 13800 tctgggatgg ggccagccccagagagagga gggcagcatg ttcggacatg ttgacttaga 13860 gatgctcaca tggtactattcagcagggag ctggcagggc ccatcagaaa ctctacagct 13920 cttcctgaga taagtttgggagttatccac acatcggtgc tcacagcagc catgggttca 13980 gataagatca ctaagtggctggagagtatg tgagagagaa ggaaatggtt gaagagcaaa 14040 tcttgcagaa caacattgaggggctaaaac cagcgaaaga ccagtcagag aggtaggagg 14100 agagccagac aggataacatgtggcagcca tgggagagga gttctgggtc aaaaaggtga 14160 gagatacaca gaagaacgagaatcaaaaca cagttccagc agaaggattg gcagatgcaa 14220 agaaacaagg gctttcttatgcaaggtagg tgtttacccg agtggcaggt atccagcatt 14280 gatgagcctg cagagccaggtgggctgcag agacaaatga gtctggactg gaagccaggc 14340 accaggactc tattctgtaaatatgggtga ggccatcaaa gccttttgaa tagaagagca 14400 ttataatgag attgtcttttagaaagattc ctataaacac taggatgaat atggaagtga 14460 acaggggcaa acccagagatgggcgaccat gaaagaggct actgctagag tccaggctga 14520 agccttgagg cccaattaaggcagaagcaa tgaggaaaga aagaaaggaa tgggtgttca 14580 aaaagtttat gatgtttaaccacaaaaagg tgaaggacaa gaggaattgg aagaaggagg 14640 gatagtggga gcgtgggagggtggtggagg tcataatgac agctgccatt atgtgtgtat 14700 aaccacacat tttatatatatatatatata tgtatataac cacattatat agtgtgtata 14760 accactgcct atgggccagacactattgca agtgaaaggc acacgtgctt ggagactgat 14820 tggatttggg gataaagcagaagggagggg aatggaaaca gaggctgaag acagacccat 14880 cagcatcaaa ccagaatccaggaggagcga gcaagcttcc ttgggctcta tgtggagatc 14940 ggtgtgtaac aggaatcacattccattctc tgtccaattc agcaccttgg agggaagcag 15000 agagaatatg agttcttcggcctgacccct gacacagagt tccttggcac catcatgatt 15060 tgcgttccca cctgggccaaggagagtgcc ccctacatgt gccgagtgaa gacactgcca 15120 ggtgagttcg gctgaaaccagagggaggtg gggctcccga attcaagtct tgactcttct 15180 ctttcctccc tgtaaccaaggtgagacttg gcaacactct agaaaaccat ggaagggtta 15240 tcagtgtgtt attgaagtcaatctttgttt atccataaaa gagttggctg atgagttatc 15300 aaagcaaatg tttgattctcaccctccagc ctatgtctgg atttcccctc tctgacaggc 15360 cttatgtgta ccttggcaaagcaaactcac cttaatatta acccaggtga gatatactgg 15420 agggaaaatc tgcaggggggaagcagagaa gataaaaaga tccctcaata tcttccaaag 15480 tcaaataata ataatggtatcatgataact agcatttctg cagtgctaac tgggaaccag 15540 gcacaaccaa gggcaagcctcatgagcagt ctatgaggta ggtattattt tcatcctcat 15600 tttacagatg aagaacctgaggttcagagt ggttaggtaa cttgcccatg gccgggcaca 15660 gtggctcaca cctgtaaaccaaacactttg ggaggccaag gcaggaggat cacttgagcc 15720 cagaagttta agaccagcctgggaaacagg gcaaaacccc atctccacaa aaaaatcaaa 15780 aacttagcta ggtatggtggcgtgcatctg tggtcccagc tacacagaag gctgaggcaa 15840 gaggatcact tgagcccaggaggttgaagc tgcaaagagc tatgtttgca ccactgcact 15900 ccagcctgga tgacagagtgagaccttgtc tcggaaaaaa aaaaaaaagt gacttgccca 15960 aggtcacaca ggtgctgagggatggaatga ggcctaattc tagaggccat gctcttacca 16020 cagagctact atagatacagttgggttttt tgtgggtttt tattgttttt tgtttttgag 16080 acagagtctt actctgtcactcaggctgga gtgcagtggc acaatctcgg ctcactacaa 16140 cttctgcttc ccagtttcaagaaattctcc cacctcagcc tcccaagtag ctgggattac 16200 aggtgtgtgc caccatgcctggctaatttt tttttttttt tttttgtatt tttagtagag 16260 acggagtttc atcctgttgctcaggctggt cttgatctcc tggcctcaag tgatccaccc 16320 acctcagcct cccaaaatgctgggattaca ggcatgagcc atcacgccca gcctagatat 16380 agttttgaat catctgctcagtgagatggt cccttcctct agcacactga aacttgattc 16440 tcatcacaag catcatgatgtttttattag tgtttctttt catagacgga gggaaacgtt 16500 gtattgctca gagctgcaggtttagaggat gaaggtgacg gcagtggatg tgatagattt 16560 ggtaaaacag tctccactttatcctcatgt tcagattcat gatgtcagta agtgatttca 16620 gttccaacca tgccaacattcagaatagag ttgagtttgc aagctggaca ttgtgtgtgg 16680 catagtggaa agagaagtaccgtgtgagga atgagaaggc acggtattag tcagatgtca 16740 gccaaccacg tgcctgggggagtcactcag cctctgggag cctgatccct catctgacaa 16800 atagggatgc tgatgcctgggctgaggtga taatcagagg ccaggcagct gccagagctg 16860 ggccctagaa aatgacactctggtcctcag gtttgttcgg aaactcatca cttttccaaa 16920 cagcccacaa gagactgatttctccacctg aatcccagtg caccaattgg caggggagct 16980 atgcctcacg gaacagccaacactggccac ttgcatgggg catgcccttc ctggggcaaa 17040 ggctgctagg gagctcagaatagaacttgg caagaggaga actagaaaaa ggaaactgac 17100 gttctggggc taagctgggagtgtttgtat gattcatgac ccccttccct ggactgtcct 17160 agataaatct cagctgctcctggcccatgg gacctggccc accagctttt tgacaatgtg 17220 aagaccactc cacccttacccacaacaccc ttctgttgcc ttactttgtc ttgcttataa 17280 tgagatacca ttttttcctaagactatagc ttttatataa atcacacttc ctggtggcta 17340 cttcttgctg acatatagaaggcaccttat ccttaaaaat atatttaaaa caattcctac 17400 atttattagg ttctactatattgccaagca aatagttgtg ccaaatgctt tacatatatt 17460 atttaatctt tattattacatgtaacatat attatttaat ctttacaaca atcctgtaag 17520 ctgggtagca tcatcctcgttttacagagg aggaactctg gctcagagag gttaagcaac 17580 ttccctgtga ccgcacagctgcttggggaa cctgattgat tcatctggtt ctgaaggctg 17640 ggctctaccc accactttgcaactttgttt acagtgagcc ccctgtactg cacacttctt 17700 cctcctactt ccatttcagagtgatgcatc ctgcccttga ctgtgtatcc tgacgtctgc 17760 attaccctca gaatcccctttacctacctc tccgtagata aacagggagg gtcacagagc 17820 attgtcccag catcttgatgacggaagagg ctctaagggc cacctcacct catatcctac 17880 tcttacagat gaggaaactgaggcttcctt gcagagtggc agggaggatg agttttgagt 17940 tacacatgta actccactgagtagctgtgt gaccttggtc aggtcactta acctctctgg 18000 gactcagctt tgttatccataggaagggag cgtccctacc cctctgtgat gtgtttgatg 18060 atgtagtgag ataacataggtagagcaccc agctctacat gatcatacag atcatggcct 18120 gtagtaggag ttcaaccactgctggctact gttattatta aagggtagac cagagagggt 18180 cgtgcagcag gaaggatgctcagcatatct gtggcagagc caggcctcta acaccctgta 18240 ttccttactc tcaggctggtggtgttttta tattccatcc agcctcccta tgggtgaaat 18300 ggcaatgtat ccaatacacacagtgtgaaa tggaaatatg tcaagccaga gaccattcca 18360 tgtccctatt ctgtcctggaacaaacagca gtggttttcc aggaatgtta tagacattcg 18420 ggtggggtct ttttcctggagtagggatgg gggtaacatg tgacaggaga cagggtttca 18480 ctgaagtggg aagctgacccagagaggccc aagctcgtgg gctaggggag gatcgaggag 18540 tgggctagaa gtagaggttccagcagtacc cggagttcac agtgatcgcc gaggtcattg 18600 gagctattga taaactctccaagctgaggc tgcacagggg ccaggaagag agggagggtg 18660 attagctggc ccctggactgcaggggtctt gactctctga gtcaccttcc ctctgttgca 18720 aactgtcagt tttaccaaaaactccaacaa cctcttcctc cataagtcct tcacaagccc 18780 cacttggact ggtgacaggaagtgtgtcct gcctggaact ggctgcaccc atcccaaaga 18840 tgctgagaca cagggagaggctcaggcact agagccctag tcatccctct cccctccatt 18900 ttagacagga cagtgccttcccaggctccc ttcttgacca agatcttccc tgatgcccag 18960 ccccttatct tggtagggaggtggagagga caggcctccc tgttttcact tctcccctcc 19020 ctctgcctct accctaccagggaggcagga ccacccttag actcaggcaa aaggggcctt 19080 tccctgcacc cagcactttagaaggccctt ctctggccct gtcctggcca ttcccctcct 19140 tgcaagatga agactccatacaacaaaggg ggcagatcca tctggatcct gtgacctcct 19200 ttctagatca cattgtgagtacctgggacc ctgaaactct ctgtacatat ggcccagagc 19260 ctgtgatccg tctaggcagtcctcttttgg gggtacttcc tcatgagggc agacactgct 19320 gacatgtagt gcagacccttggcccaaagg atgaccaaca ggtggctatt tggaggggac 19380 atggatgagc tcgaccatgagggctggggc atctatacac atgtgcgtga gggccctggc 19440 agtgtgggat ggaactgggctgggctgggc tgggctgggg gcctgcagtg cgctgcgggc 19500 tttccctgta cctccccacttcagcacagt acctagagaa gtccaagaac tctgaattaa 19560 aatatggcat cccaggtggttgtaaaaaat ctttttgtta aggtggaaac ctcgaacatt 19620 ttatttattt aacagtgtaaaaatataatt tgtaaattct gactatttag atgtacagta 19680 tatggacttc catttgtgatcttgcattct gacggtagga gaggccttgc cagcccagcc 19740 agagagggtt ttcagcatccacatgtgggg ccagacccaa ccgggcccag gcagagccca 19800 gcctcacgct tccttctctggcccctttgg tttcccagac cggacatgga cctactcctt 19860 ctccggagcc ttcctgttctccatgggctt cctcgtcgca gtactctgct acctgagcta 19920 cagatatgtc accaagccgcctgcacctcc caactccctg gtgagccatc cccccagggt 19980 gggagggaga ggtccaggggagggcctggg ctccgaggtc cccagttgtc cccaaaatgc 20040 agggatccac agacaaagcaggagatgggc ctgctctctc cattcacgga gctcacagtc 20100 ccagctaaag tcaacggacgtgtagcccag caggtctcag gacacattga agtggggtgg 20160 gtgatcagga atagtagacatggaataaaa aacccacatg gcttggaagt gtccagcacc 20220 aggagggtag tgtgggacttgagtgagcag caaggcaacg agggaggaat ggtgggcgcc 20280 aggagagccg tggcactcatgccctgtcta aatgggcagc cgggcaccac catgtgggaa 20340 tgcaggccca gcactgcctgatcttcatgt ttttcaagag aagccagagt ttcagattat 20400 attgtggaat ctcccaattttcaaatattc agaactaatt ttttaaacag attttttttt 20460 ttttgagatg gagtctccctctgtcaccca ggctggagtg cagtggcatg atctcgtctc 20520 agcacaacct ccacctcccgagttcaagcg attctcctgc ctcatcctcc caagtagctg 20580 ggattacagg catgcaccaccaccacacct ggctaatttt tgtattttta gtagagacgg 20640 gttttaccat gttggccaggctggtctcaa actcctgact tcaggtgatc tacccacctc 20700 ggcctcccaa agtgctgggattacaagcgt gagccactgc acctggccat tttaaacaga 20760 aatttttaaa tggcaaaactaattgttaag tggaaagccc tttgttacag ttcttttaga 20820 attgctgtag caagtgaagaccagaaccac cccccaccca aaaaataaaa ccacccccca 20880 cccaaaaaat aaaaataaaatggaaagccc tacatcagcc acacaaatca catcagcagc 20940 ccccacctga cgccagtgccgcctctgcta gagcagcaag gccactgcag aagccatgag 21000 ctgagccacc tgaggcacccggagcctgct cttgcccatc ggagggacag aggagctctg 21060 agggagtttt gctcaatgtgcaagtccaga tgaagcattt tctgtaaagc tctcccggtc 21120 tctcctggct ccttcttttcccaccctgac cctgtattag gccctcaggg acccgtgggc 21180 caagcaacaa tgcgggccaccacctcgaga tcatgcccca aggcaacccc aaatcctggt 21240 catctactcc ctggattatccgcctcctgg agaaaccaga gcccagatct ctctccctga 21300 gataacccag gctttagaaccaaagagctg agagaccctg tcccttcaga gaggcacttg 21360 cacctagagg agtctctgggaagcagatgg ggatatggga cagacgcatc ttgaaaaagc 21420 ccccagatgc ctccctatggaggacctcac ccacccacat caccagtagg gagcttggga 21480 cttaccctaa ccacaggggggtgactgttg tcgtccctgc acagaacgtc cagcgagtcc 21540 tgactttcca gccgctgcgcttcatccagg agcacgtcct gatccctgtc tttgacctca 21600 gcggccccag cagtctggcccagcctgtcc agtactccca gatcagggtg tctggaccca 21660 gggagcccgc aggagctccacagcggcata gcctgtccga gatcacctac ttagggcagc 21720 cagacatctc catcctccagccctccaacg tgccacctcc ccagatcctc tccccactgt 21780 cctatgcccc aaacgctgcccctgaggtcg ggcccccatc ctatgcacct caggtgaccc 21840 ccgaagctca attcccattctacgccccac aggccatctc taaggtccag ccttcctcct 21900 atgcccctca agccactccggacagctggc ctccctccta tggggtatgc atggaaggtt 21960 ctggcaaaga ctcccccactgggacacttt ctagtcctaa acaccttagg cctaaaggtc 22020 agcttcagaa agagccaccagctggaagct gcatgttagg tggcctttct ctgcaggagg 22080 tgacctcctt ggctatggaggaatcccaag aagcaaaatc attgcaccag cccctgggga 22140 tttgcacaga cagaacatctgacccaaatg tgctacacag tggggaggaa gggacaccac 22200 agtacctaaa gggccagctccccctcctct cctcagtcca gatcgagggc caccccatgt 22260 ccctcccttt gcaacctccttcccgtccat gttccccctc ggaccaaggt ccaagtccct 22320 ggggcctgct ggagtcccttgtgtgtccca aggatgaagc caagagccca gcccctgaga 22380 cctcagacct ggagcagcccacagaactgg attctctttt cagaggcctg gccctgactg 22440 tgcagtggga gtcctgaggggaatgggaaa ggcttggtgc ttcctccctg tccctaccca 22500 gtgtcacatc cttggctgtcaatcccatgc ctgcccacgc cacacactct gcgatctggc 22560 ctcagacggg tgcccttgagagaagcagag ggagtggcat gcagggcccc tgccatgggt 22620 gcgctcctca ccggagcaaagcagcatgat aaggactgca gcgggggagc tctggggagc 22680 agcttgtgta gacaagcgcgtgctcgctga gccctgcaag gcagaaatga cagtgcaagg 22740 aggaaatgca gggaaactcccgaggtccag agccccacct cctaacacca tggattcaaa 22800 gtgctcaggg aatttgcctctccttgcccc attcctggcc agtttcacaa tctagctcga 22860 cagagcatga ggcccctgcctcttctgtca ttgttcaaag gtgggaagag agcctggaaa 22920 agaaccaggc ctggaaaagaaccagaagga ggctgggcag aaccagaaca acctgcactt 22980 ctgccaaggc cagggccagcaggacggcag gactctaggg aggggtgtgg cctgcagctc 23040 attcccagcc agggcaactgcctgacgttg cacgatttca gcttcattcc tctgatagaa 23100 caaagcgaaa tgcaggtccaccagggaggg agacacacaa gccttttctg caggcaggag 23160 tttcagaccc tatcctgagaatggggtttg aaaggaaggt gagggctgtg gcccctggac 23220 gggtacaata acacactgtactgatgtcac aactttgcaa gctctgcctt gggttcagcc 23280 catctgggct caaattccagcctcaccact cacaagctgt gtgacttcaa acaaatgaaa 23340 tcagtgccca gaacctcggtttcctcatct gtaatgtggg gatcataaca cctacctcat 23400 ggagttgtgg tgaagatgaaatgaagtcat gtctttaaag tgcttaatag tgcctggtac 23460 atgggcagtg cccaataaacggtagctatt tcctgttgtg attttttttt taaactacgt 23520 tacacaagga gtgaccccctcccccaattc ggattggctt cagacacacc tggcattttc 23580 tcatgggctt aaatgacctggatttcctca ggacaggcaa agccagaggg gtctggggtg 23640 ggaaagagga gggactgcagggccttctag agacaagagt tcagagacaa atgatgtgga 23700 accttctccc tgagatcgtaggcagaagcc attgtgggtg gcagtggtaa tctcagacgg 23760 cctctgtcca tccagcctccctgatccccc acagaggcag tgttgcacac tctgtgctct 23820 cagactgctg ccataccctccgtatcactg catctcagac gacccctacc atgaggaagc 23880 caaagtccag agggagaggggacctacccg agatcccact attgaggctg tgacagagtt 23940 ggagtgcaaa cctagggctttttgtcctca tcgagagagg aagctttcca cacctgcttg 24000 g 24001 <210> SEQ IDNO 5 <211> LENGTH: 17 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: PCR Primer <400>SEQUENCE: 5 tacccccacg ccaatcc 17 <210> SEQ ID NO 6 <211> LENGTH: 25<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223>OTHER INFORMATION: PCR Primer <400> SEQUENCE: 6 ggtagaacag gtcatggaagatgtc 25 <210> SEQ ID NO 7 <211> LENGTH: 21 <212> TYPE: DNA <213>ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION:PCR Probe <400> SEQUENCE: 7 caggcgatgg ccaccggcta a 21 <210> SEQ ID NO 8<211> LENGTH: 19 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: PCR Primer <400> SEQUENCE: 8gaaggtgaag gtcggagtc 19 <210> SEQ ID NO 9 <211> LENGTH: 20 <212> TYPE:DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: PCR Primer <400> SEQUENCE: 9 gaagatggtg atgggatttc 20 <210>SEQ ID NO 10 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: PCR Probe <400>SEQUENCE: 10 caagcttccc gttctcagcc 20 <210> SEQ ID NO 11 <211> LENGTH:2795 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220> FEATURE: <221>NAME/KEY: CDS <222> LOCATION: (24)...(1748) <400> SEQUENCE: 11gggagggctc tgtgccagcc ccg atg agg acg ctg ctg acc atc ttg act gtg 53 MetArg Thr Leu Leu Thr Ile Leu Thr Val 1 5 10 gga tcc ctg gct gct cac gcccct gag gac ccc tcg gat ctg ctc cag 101 Gly Ser Leu Ala Ala His Ala ProGlu Asp Pro Ser Asp Leu Leu Gln 15 20 25 cac gtg aaa ttc cag tcc agc aacttt gaa aac atc ctg acg tgg gac 149 His Val Lys Phe Gln Ser Ser Asn PheGlu Asn Ile Leu Thr Trp Asp 30 35 40 agc ggg cca gag ggc acc cca gac acggtc tac agc atc gag tat aag 197 Ser Gly Pro Glu Gly Thr Pro Asp Thr ValTyr Ser Ile Glu Tyr Lys 45 50 55 acg tac gga gag agg gac tgg gtg gca aagaag ggc tgt cag cgg atc 245 Thr Tyr Gly Glu Arg Asp Trp Val Ala Lys LysGly Cys Gln Arg Ile 60 65 70 acc cgg aag tcc tgc aac ctg acg gtg gag acgggc aac ctc acg gag 293 Thr Arg Lys Ser Cys Asn Leu Thr Val Glu Thr GlyAsn Leu Thr Glu 75 80 85 90 ctc tac tat gcc agg gtc acc gct gtc agt gcggga ggc cgg tca gcc 341 Leu Tyr Tyr Ala Arg Val Thr Ala Val Ser Ala GlyGly Arg Ser Ala 95 100 105 acc aag atg act gac agg ttc agc tct ctg cagcac act acc ctc aag 389 Thr Lys Met Thr Asp Arg Phe Ser Ser Leu Gln HisThr Thr Leu Lys 110 115 120 cca cct gat gtg acc tgt atc tcc aaa gtg agatcg att cag atg att 437 Pro Pro Asp Val Thr Cys Ile Ser Lys Val Arg SerIle Gln Met Ile 125 130 135 gtt cat cct acc ccc acg cca atc cgt gca ggcgat ggc cac cgg cta 485 Val His Pro Thr Pro Thr Pro Ile Arg Ala Gly AspGly His Arg Leu 140 145 150 acc ctg gaa gac atc ttc cat gac ctg ttc taccac tta gag ctc cag 533 Thr Leu Glu Asp Ile Phe His Asp Leu Phe Tyr HisLeu Glu Leu Gln 155 160 165 170 gtc aac cgc acc tac caa atg cac ctt ggaggg aag cag aga gaa tat 581 Val Asn Arg Thr Tyr Gln Met His Leu Gly GlyLys Gln Arg Glu Tyr 175 180 185 gag ttc ttc ggc ctg acc cct gac aca gagttc ctt ggc acc atc atg 629 Glu Phe Phe Gly Leu Thr Pro Asp Thr Glu PheLeu Gly Thr Ile Met 190 195 200 att tgc gtt ccc acc tgg gcc aag gag agtgcc ccc tac atg tgc cga 677 Ile Cys Val Pro Thr Trp Ala Lys Glu Ser AlaPro Tyr Met Cys Arg 205 210 215 gtg aag aca ctg cca gac cgg aca tgg acctac tcc ttc tcc gga gcc 725 Val Lys Thr Leu Pro Asp Arg Thr Trp Thr TyrSer Phe Ser Gly Ala 220 225 230 ttc ctg ttc tcc atg ggc ttc ctc gtc gcagta ctc tgc tac ctg agc 773 Phe Leu Phe Ser Met Gly Phe Leu Val Ala ValLeu Cys Tyr Leu Ser 235 240 245 250 tac aga tat gtc acc aag ccg cct gcacct ccc aac tcc ctg aac gtc 821 Tyr Arg Tyr Val Thr Lys Pro Pro Ala ProPro Asn Ser Leu Asn Val 255 260 265 cag cga gtc ctg act ttc cag ccg ctgcgc ttc atc cag gag cac gtc 869 Gln Arg Val Leu Thr Phe Gln Pro Leu ArgPhe Ile Gln Glu His Val 270 275 280 ctg atc cct gtc ttt gac ctc agc ggcccc agc agt ctg gcc cag cct 917 Leu Ile Pro Val Phe Asp Leu Ser Gly ProSer Ser Leu Ala Gln Pro 285 290 295 gtc cag tac tcc cag atc agg gtg tctgga ccc agg gag ccc gca gga 965 Val Gln Tyr Ser Gln Ile Arg Val Ser GlyPro Arg Glu Pro Ala Gly 300 305 310 gct cca cag cgg cat agc ctg tcc gagatc acc tac tta ggg cag cca 1013 Ala Pro Gln Arg His Ser Leu Ser Glu IleThr Tyr Leu Gly Gln Pro 315 320 325 330 gac atc tcc atc ctc cag ccc tccaac gtg cca cct ccc cag atc ctc 1061 Asp Ile Ser Ile Leu Gln Pro Ser AsnVal Pro Pro Pro Gln Ile Leu 335 340 345 tcc cca ctg tcc tat gcc cca aacgct gcc cct gag gtc ggg ccc cca 1109 Ser Pro Leu Ser Tyr Ala Pro Asn AlaAla Pro Glu Val Gly Pro Pro 350 355 360 tcc tat gca cct cag gtg acc cccgaa gct caa ttc cca ttc tac gcc 1157 Ser Tyr Ala Pro Gln Val Thr Pro GluAla Gln Phe Pro Phe Tyr Ala 365 370 375 cca cag gcc atc tct aag gtc cagcct tcc tcc tat gcc cct caa gcc 1205 Pro Gln Ala Ile Ser Lys Val Gln ProSer Ser Tyr Ala Pro Gln Ala 380 385 390 act ccg gac agc tgg cct ccc tcctat ggg gta tgc atg gaa ggt tct 1253 Thr Pro Asp Ser Trp Pro Pro Ser TyrGly Val Cys Met Glu Gly Ser 395 400 405 410 ggc aaa gac tcc ccc act gggaca ctt tct agt cct aaa cac ctt agg 1301 Gly Lys Asp Ser Pro Thr Gly ThrLeu Ser Ser Pro Lys His Leu Arg 415 420 425 cct aaa ggt cag ctt cag aaagag cca cca gct gga agc tgc atg tta 1349 Pro Lys Gly Gln Leu Gln Lys GluPro Pro Ala Gly Ser Cys Met Leu 430 435 440 ggt ggc ctt tct ctg cag gaggtg acc tcc ttg gct atg gag gaa tcc 1397 Gly Gly Leu Ser Leu Gln Glu ValThr Ser Leu Ala Met Glu Glu Ser 445 450 455 caa gaa gca aaa tca ttg caccag ccc ctg ggg att tgc aca gac aga 1445 Gln Glu Ala Lys Ser Leu His GlnPro Leu Gly Ile Cys Thr Asp Arg 460 465 470 aca tct gac cca aat gtg ctacac agt ggg gag gaa ggg aca cca cag 1493 Thr Ser Asp Pro Asn Val Leu HisSer Gly Glu Glu Gly Thr Pro Gln 475 480 485 490 tac cta aag ggc cag ctcccc ctc ctc tcc tca gtc cag atc gag ggc 1541 Tyr Leu Lys Gly Gln Leu ProLeu Leu Ser Ser Val Gln Ile Glu Gly 495 500 505 cac ccc atg tcc ctc cctttg caa cct cct tcc ggt cca tgt tcc ccc 1589 His Pro Met Ser Leu Pro LeuGln Pro Pro Ser Gly Pro Cys Ser Pro 510 515 520 tcg gac caa ggt cca agtccc tgg ggc ctg ctg gag tcc ctt gtg tgt 1637 Ser Asp Gln Gly Pro Ser ProTrp Gly Leu Leu Glu Ser Leu Val Cys 525 530 535 ccc aag gat gaa gcc aagagc cca gcc cct gag acc tca gac ctg gag 1685 Pro Lys Asp Glu Ala Lys SerPro Ala Pro Glu Thr Ser Asp Leu Glu 540 545 550 cag ccc aca gaa ctg gattct ctt ttc aga ggc ctg gcc ctg act gtg 1733 Gln Pro Thr Glu Leu Asp SerLeu Phe Arg Gly Leu Ala Leu Thr Val 555 560 565 570 cag tgg gag tcc tgaggggaatggg aaaggcttgg tgcttcctcc ctgtccctac 1788 Gln Trp Glu Serccagtgtcac atccttggct gtcaatccca tgcctgccca tgccacacac tctgcgatct 1848ggcctcagac gggtgccctt gagagaagca gagggagtgg catgcagggc ccctgccatg 1908ggtgcgctcc tcaccggaac aaagcagcat gataaggact gcagcggggg agctctgggg 1968agcagcttgt gtagacaagc gcgtgctcgc tgagccctgc aaggcagaaa tgacagtgca 2028aggaggaaat gcagggaaac tcccgaggtc cagagcccca cctcctaaca ccatggattc 2088aaagtgctca gggaatttgc ctctccttgc cccattcctg gccagtttca caatctagct 2148cgacagagca tgaggcccct gcctcttctg tcattgttca aaggtgggaa gagagcctgg 2208aaaagaacca ggcctggaaa agaaccagaa ggaggctggg cagaaccaga acaacctgca 2268cttctgccaa ggccagggcc agcaggacgg caggactcta gggaggggtg tggcctgcag 2328ctcattccca gccagggcaa ctgcctgacg ttgcacgatt tcagcttcat tcctctgata 2388gaacaaagcg aaatgcaggt ccaccaggga gggagacaca caagcctttt ctgcaggcag 2448gagtttcaga ccctatcctg agaatggggt ttgaaaggaa ggtgagggct gtggcccctg 2508gacgggtaca ataacacact gtactgatgt cacaactttg caagctctgc cttgggttca 2568gcccatctgg gctcaaattc cagcctcacc actcacaagc tgtgtgactt caaacaaatg 2628aaatcagtgc ccagaacctc ggtttcctca tctgtaatgt ggggatcata acacctacct 2688catggagttg tggtgaagat gaaatgaagt catgtcttta aagtgcttaa tagtgcctgg 2748tacatgggca gtgcccaata aacggtagct atttaaaaaa aaaaaaa 2795 <210> SEQ ID NO12 <220> FEATURE: <400> SEQUENCE: 12 000 <210> SEQ ID NO 13 <211>LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220>FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400>SEQUENCE: 13 gggctggcac agagccctcc 20 <210> SEQ ID NO 14 <211> LENGTH:20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 14agccagggat cccacagtca 20 <210> SEQ ID NO 15 <211> LENGTH: 20 <212> TYPE:DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 15 cacgtgctggagcagatccg 20 <210> SEQ ID NO 16 <211> LENGTH: 20 <212> TYPE: DNA <213>ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION:Antisense Oligonucleotide <400> SEQUENCE: 16 aagttgctgg actggaattt 20<210> SEQ ID NO 17 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 17 ctctccgtac gtcttatact 20 <210> SEQ IDNO 18 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 18 atccgctgac agcccttctt 20 <210> SEQ IDNO 19 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 19 accgtcaggt tgcaggactt 20 <210> SEQ IDNO 20 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 20 tagtagagct ccgtgaggtt 20 <210> SEQ IDNO 21 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 21 ttgagggtag tgtgctgcag 20 <210> SEQ IDNO 22 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 22 tagaacaggt catggaagat 20 <210> SEQ IDNO 23 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 23 gcttccctcc aaggtgcatt 20 <210> SEQ IDNO 24 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 24 ggccgaagaa ctcatattct 20 <210> SEQ IDNO 25 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 25 gatggtgcca aggaactctg 20 <210> SEQ IDNO 26 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 26 ggtctggcag tgtcttcact 20 <210> SEQ IDNO 27 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 27 catgtccggt ctggcagtgt 20 <210> SEQ IDNO 28 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 28 gagtaggtcc atgtccggtc 20 <210> SEQ IDNO 29 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 29 acaggaaggc tccggagaag 20 <210> SEQ IDNO 30 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 30 ggagaacagg aaggctccgg 20 <210> SEQ IDNO 31 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 31 aagcccatgg agaacaggaa 20 <210> SEQ IDNO 32 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 32 tcaggtagca gagtactgcg 20 <210> SEQ IDNO 33 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 33 catatctgta gctcaggtag 20 <210> SEQ IDNO 34 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 34 cgctgaggtc aaagacaggg 20 <210> SEQ IDNO 35 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 35 agacaccctg atctgggagt 20 <210> SEQ IDNO 36 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 36 agctcctgcg ggctccctgg 20 <210> SEQ IDNO 37 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 37 tgtggagctc ctgcgggctc 20 <210> SEQ IDNO 38 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 38 caggctatgc cgctgtggag 20 <210> SEQ IDNO 39 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 39 aagtaggtga tctcggacag 20 <210> SEQ IDNO 40 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 40 gtctggctgc cctaagtagg 20 <210> SEQ IDNO 41 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 41 ggtcacctga ggtgcatagg 20 <210> SEQ IDNO 42 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 42 agctgtccgg agtggcttga 20 <210> SEQ IDNO 43 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 43 ccccatagga gggaggccag 20 <210> SEQ IDNO 44 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 44 tgcatacccc ataggaggga 20 <210> SEQ IDNO 45 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 45 aaccttccat gcatacccca 20 <210> SEQ IDNO 46 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 46 agtctttgcc agaaccttcc 20 <210> SEQ IDNO 47 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 47 ttaggactag aaagtgtccc 20 <210> SEQ IDNO 48 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 48 agcttccagc tggtggctct 20 <210> SEQ IDNO 49 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 49 cctaacatgc agcttccagc 20 <210> SEQ IDNO 50 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 50 tcctgcagag aaaggccacc 20 <210> SEQ IDNO 51 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 51 ccatagccaa ggaggtcacc 20 <210> SEQ IDNO 52 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 52 ctgtctgtgc aaatccccag 20 <210> SEQ IDNO 53 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 53 tgtagcacat ttgggtcaga 20 <210> SEQ IDNO 54 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 54 ctggcccttt aggtactgtg 20 <210> SEQ IDNO 55 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 55 gtggccctcg atctggactg 20 <210> SEQ IDNO 56 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 56 gggactccag caggccccag 20 <210> SEQ IDNO 57 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 57 cttggcttca tccttgggac 20 <210> SEQ IDNO 58 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 58 gctgctccag gtctgaggtc 20 <210> SEQ IDNO 59 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 59 ggccaggcct ctgaaaagag 20 <210> SEQ IDNO 60 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 60 tcccactgca cagtcagggc 20 <210> SEQ IDNO 61 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 61 ttcccattcc cctcaggact 20 <210> SEQ IDNO 62 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 62 ctccctctgc ttctctcaag 20 <210> SEQ IDNO 63 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 63 tccggtgagg agcgcaccca 20 <210> SEQ IDNO 64 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 64 gcgcttgtct acacaagctg 20 <210> SEQ IDNO 65 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 65 ggagtttccc tgcatttcct 20 <210> SEQ IDNO 66 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 66 ttccctgagc actttgaatc 20 <210> SEQ IDNO 67 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 67 tcgagctaga ttgtgaaact 20 <210> SEQ IDNO 68 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 68 cttttccagg cctggttctt 20 <210> SEQ IDNO 69 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 69 ttctggttct gcccagcctc 20 <210> SEQ IDNO 70 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 70 cagaagtgca ggttgttctg 20 <210> SEQ IDNO 71 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 71 ctgggaatga gctgcaggcc 20 <210> SEQ IDNO 72 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 72 caggcagttg ccctggctgg 20 <210> SEQ IDNO 73 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 73 ttgttctatc agaggaatga 20 <210> SEQ IDNO 74 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 74 tccctccctg gtggacctgc 20 <210> SEQ IDNO 75 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 75 ttctcaggat agggtctgaa 20 <210> SEQ IDNO 76 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 76 gggctgaacc caaggcagag 20 <210> SEQ IDNO 77 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 77 atcttcacca caactccatg 20 <210> SEQ IDNO 78 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 78 atgacttcat ttcatcttca 20 <210> SEQ IDNO 79 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 79 cccatgtacc aggcactatt 20 <210> SEQ IDNO 80 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 80 accgtttatt gggcactgcc 20 <210> SEQ IDNO 81 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 81 aaatagctac cgtttattgg 20 <210> SEQ IDNO 82 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 82 cattccataa atgtcaccac 20 <210> SEQ IDNO 83 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 83 agaagcctac gtcttatact 20 <210> SEQ IDNO 84 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 84 ctctccgtac ctgcaggtca 20 <210> SEQ IDNO 85 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 85 tttaactgac tcacagttcc 20 <210> SEQ IDNO 86 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 86 cctcatttac cctgaatcta 20 <210> SEQ IDNO 87 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 87 ccgaactcac ctggcagtgt 20 <210> SEQ IDNO 88 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 88 gatggctcac cagggagttg 20 <210> SEQ IDNO 89 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 89 ccaccccact tcaatgtgtc 20 <210> SEQ IDNO 90 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 90 ctcccttggc ctctactctg 20 <210> SEQ IDNO 91 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220>FEATURE: <400> SEQUENCE: 91 tgactgtggg atccctggct 20 <210> SEQ ID NO 92<211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220>FEATURE: <400> SEQUENCE: 92 cggatctgct ccagcacgtg 20 <210> SEQ ID NO 93<211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220>FEATURE: <400> SEQUENCE: 93 aaattccagt ccagcaactt 20 <210> SEQ ID NO 94<211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220>FEATURE: <400> SEQUENCE: 94 agtataagac gtacggagag 20 <210> SEQ ID NO 95<211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220>FEATURE: <400> SEQUENCE: 95 aagaagggct gtcagcggat 20 <210> SEQ ID NO 96<211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220>FEATURE: <400> SEQUENCE: 96 aagtcctgca acctgacggt 20 <210> SEQ ID NO 97<211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220>FEATURE: <400> SEQUENCE: 97 aacctcacgg agctctacta 20 <210> SEQ ID NO 98<211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220>FEATURE: <400> SEQUENCE: 98 aatgcacctt ggagggaagc 20 <210> SEQ ID NO 99<211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220>FEATURE: <400> SEQUENCE: 99 agaatatgag ttcttcggcc 20 <210> SEQ ID NO 100<211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220>FEATURE: <400> SEQUENCE: 100 cagagttcct tggcaccatc 20 <210> SEQ ID NO101 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220>FEATURE: <400> SEQUENCE: 101 ccggagcctt cctgttctcc 20 <210> SEQ ID NO102 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220>FEATURE: <400> SEQUENCE: 102 ttcctgttct ccatgggctt 20 <210> SEQ ID NO103 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220>FEATURE: <400> SEQUENCE: 103 cgcagtactc tgctacctga 20 <210> SEQ ID NO104 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220>FEATURE: <400> SEQUENCE: 104 ctacctgagc tacagatatg 20 <210> SEQ ID NO105 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220>FEATURE: <400> SEQUENCE: 105 ccctgtcttt gacctcagcg 20 <210> SEQ ID NO106 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220>FEATURE: <400> SEQUENCE: 106 actcccagat cagggtgtct 20 <210> SEQ ID NO107 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220>FEATURE: <400> SEQUENCE: 107 ccagggagcc cgcaggagct 20 <210> SEQ ID NO108 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220>FEATURE: <400> SEQUENCE: 108 ctccacagcg gcatagcctg 20 <210> SEQ ID NO109 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220>FEATURE: <400> SEQUENCE: 109 cctacttagg gcagccagac 20 <210> SEQ ID NO110 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220>FEATURE: <400> SEQUENCE: 110 cctatgcacc tcaggtgacc 20 <210> SEQ ID NO111 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220>FEATURE: <400> SEQUENCE: 111 tcaagccact ccggacagct 20 <210> SEQ ID NO112 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220>FEATURE: <400> SEQUENCE: 112 tccctcctat ggggtatgca 20 <210> SEQ ID NO113 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220>FEATURE: <400> SEQUENCE: 113 tggggtatgc atggaaggtt 20 <210> SEQ ID NO114 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220>FEATURE: <400> SEQUENCE: 114 ggaaggttct ggcaaagact 20 <210> SEQ ID NO115 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220>FEATURE: <400> SEQUENCE: 115 gctggaagct gcatgttagg 20 <210> SEQ ID NO116 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220>FEATURE: <400> SEQUENCE: 116 ggtggccttt ctctgcagga 20 <210> SEQ ID NO117 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220>FEATURE: <400> SEQUENCE: 117 ggtgacctcc ttggctatgg 20 <210> SEQ ID NO118 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220>FEATURE: <400> SEQUENCE: 118 ctggggattt gcacagacag 20 <210> SEQ ID NO119 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220>FEATURE: <400> SEQUENCE: 119 tctgacccaa atgtgctaca 20 <210> SEQ ID NO120 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220>FEATURE: <400> SEQUENCE: 120 cacagtacct aaagggccag 20 <210> SEQ ID NO121 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220>FEATURE: <400> SEQUENCE: 121 cagtccagat cgagggccac 20 <210> SEQ ID NO122 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220>FEATURE: <400> SEQUENCE: 122 ctggggcctg ctggagtccc 20 <210> SEQ ID NO123 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220>FEATURE: <400> SEQUENCE: 123 gacctcagac ctggagcagc 20 <210> SEQ ID NO124 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220>FEATURE: <400> SEQUENCE: 124 ctcttttcag aggcctggcc 20 <210> SEQ ID NO125 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220>FEATURE: <400> SEQUENCE: 125 gccctgactg tgcagtggga 20 <210> SEQ ID NO126 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220>FEATURE: <400> SEQUENCE: 126 agtcctgagg ggaatgggaa 20 <210> SEQ ID NO127 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220>FEATURE: <400> SEQUENCE: 127 cttgagagaa gcagagggag 20 <210> SEQ ID NO128 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220>FEATURE: <400> SEQUENCE: 128 cagcttgtgt agacaagcgc 20 <210> SEQ ID NO129 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220>FEATURE: <400> SEQUENCE: 129 aggaaatgca gggaaactcc 20 <210> SEQ ID NO130 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220>FEATURE: <400> SEQUENCE: 130 gattcaaagt gctcagggaa 20 <210> SEQ ID NO131 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220>FEATURE: <400> SEQUENCE: 131 agtttcacaa tctagctcga 20 <210> SEQ ID NO132 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220>FEATURE: <400> SEQUENCE: 132 gaggctgggc agaaccagaa 20 <210> SEQ ID NO133 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220>FEATURE: <400> SEQUENCE: 133 cagaacaacc tgcacttctg 20 <210> SEQ ID NO134 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220>FEATURE: <400> SEQUENCE: 134 ccagccaggg caactgcctg 20 <210> SEQ ID NO135 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220>FEATURE: <400> SEQUENCE: 135 tcattcctct gatagaacaa 20 <210> SEQ ID NO136 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220>FEATURE: <400> SEQUENCE: 136 ctctgccttg ggttcagccc 20 <210> SEQ ID NO137 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220>FEATURE: <400> SEQUENCE: 137 tgaagatgaa atgaagtcat 20 <210> SEQ ID NO138 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220>FEATURE: <400> SEQUENCE: 138 aatagtgcct ggtacatggg 20 <210> SEQ ID NO139 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220>FEATURE: <400> SEQUENCE: 139 ggcagtgccc aataaacggt 20 <210> SEQ ID NO140 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220>FEATURE: <400> SEQUENCE: 140 agtataagac gtaggcttct 20 <210> SEQ ID NO141 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220>FEATURE: <400> SEQUENCE: 141 tgacctgcag gtacggagag 20 <210> SEQ ID NO142 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220>FEATURE: <400> SEQUENCE: 142 ggaactgtga gtcagttaaa 20 <210> SEQ ID NO143 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220>FEATURE: <400> SEQUENCE: 143 acactgccag gtgagttcgg 20 <210> SEQ ID NO144 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220>FEATURE: <400> SEQUENCE: 144 cagagtagag gccaagggag 20

What is claimed is:
 1. A compound 8 to 80 nucleobases in length targetedto a nucleic acid molecule encoding interleukin 22 receptor, whereinsaid compound specifically hybridizes with said nucleic acid moleculeencoding interleukin 22 receptor (SEQ ID NO: 4) and inhibits theexpression of interleukin 22 receptor.
 2. The compound of claim 1comprising 12 to 50 nucleobases in length.
 3. The compound of claim 2comprising 15 to 30 nucleobases in length.
 4. The compound of claim 1comprising an oligonucleotide.
 5. The compound of claim 4 comprising anantisense oligonucleotide.
 6. The compound of claim 4 comprising a DNAoligonucleotide.
 7. The compound of claim 4 comprising an RNAoligonucleotide.
 8. The compound of claim 4 comprising a chimericoligonucleotide.
 9. The compound of claim 4 wherein at least a portionof said compound hybridizes with RNA to form an oligonucleotide-RNAduplex.
 10. The compound of claim 1 having at least 70% complementaritywith a nucleic acid molecule encoding interleukin 22 receptor (SEQ IDNO: 4) said compound specifically hybridizing to and inhibiting theexpression of interleukin 22 receptor.
 11. The compound of claim 1having at least 80% complementarity with a nucleic acid moleculeencoding interleukin 22 receptor (SEQ ID NO: 4) said compoundspecifically hybridizing to and inhibiting the expression of interleukin22 receptor.
 12. The compound of claim 1 having at least 90%complementarity with a nucleic acid molecule encoding interleukin 22receptor (SEQ ID NO: 4) said compound specifically hybridizing to andinhibiting the expression of interleukin 22 receptor.
 13. The compoundof claim 1 having at least 95% complementarity with a nucleic acidmolecule encoding interleukin 22 receptor (SEQ ID NO: 4) said compoundspecifically hybridizing to and inhibiting the expression of interleukin22 receptor.
 14. The compound of claim 1 having at least one modifiedinternucleoside linkage, sugar moiety, or nucleobase.
 15. The compoundof claim 1 having at least one 2′-0-methoxyethyl sugar moiety.
 16. Thecompound of claim 1 having at least one phosphorothioate internucleosidelinkage.
 17. The compound of claim 1 having at least one5-methylcytosine.
 18. A method of inhibiting the expression ofinterleukin 22 receptor in cells or tissues comprising contacting saidcells or tissues with the compound of claim 1 so that expression ofinterleukin 22 receptor is inhibited.
 19. A method of screening for amodulator of interleukin 22 receptor, the method comprising the stepsof: a. contacting a preferred target segment of a nucleic acid moleculeencoding interleukin 22 receptor with one or more candidate modulatorsof interleukin 22 receptor, and b. identifying one or more modulators ofinterleukin 22 receptor expression which modulate the expression ofinterleukin 22 receptor.
 20. The method of claim 21 wherein themodulator of interleukin 22 receptor expression comprises anoligonucleotide, an antisense oligonucleotide, a DNA oligonucleotide, anRNA oligonucleotide, an RNA oligonucleotide having at least a portion ofsaid RNA oligonucleotide capable of hybridizing with RNA to form anoligonucleotide-RNA duplex, or a chimeric oligonucleotide.
 21. Adiagnostic method for identifying a disease state comprising identifyingthe presence of interleukin 22 receptor in a sample using at least oneof the primers comprising SEQ ID NOs 5 or 6, or the probe comprising SEQID NO
 7. 22. A kit or assay device comprising the compound of claim 1.23. A method of treating an animal having a disease or conditionassociated with interleukin 22 receptor comprising administering to saidanimal a therapeutically or prophylactically effective amount of thecompound of claim 1 so that expression of interleukin 22 receptor isinhibited.
 24. The method of claim 23 wherein the disease or conditionis an autoimmune disorder.